Substituted pyrrolidine and related compounds

ABSTRACT

This invention is directed to compounds of formula I: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1 –R 5  and a-e are as defined in the specification; or pharmaceutically-acceptable salt or solvate or stereoisomer thereof. The invention also directed to pharmaceutical compositions containing such compounds; processes and intermediates useful for preparing such compounds; and methods for treating disease conditions mediated by muscarinic receptors using such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/478,456, filed on Jun. 13, 2003; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to substituted pyrrolidine and relatedcompounds having muscarinic receptor antagonist or anticholinergicactivity. This invention is also directed to pharmaceutical compositionscomprising such compounds; methods of using such compounds to treatmedical conditions mediated by muscarinic receptors; and processes andintermediates useful for preparing such compounds.

2. State of the Art

Pulmonary disorders, such as chronic obstructive pulmonary disease(COPD) and asthma, afflict many millions of people worldwide and suchdisorders are a leading cause of morbidity and mortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, urinary retention, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for new muscarinic receptor antagonists havinghigh potency and reduced systemic side effects when administered byinhalation. Additionally, a need exists for inhaled muscarinic receptorantagonists having a long duration of action thereby allowing foronce-daily or even once-weekly dosing. Such compounds are expected to beparticularly effective for treating pulmonary disorders, such as COPDand asthma, while reducing or eliminating side effects, such asdry-mouth.

SUMMARY OF THE INVENTION

The present invention provides novel substituted pyrrolidine and relatedcompounds which have muscarinic receptor antagonist or anticholinergicactivity. Among other properties, compounds of this invention have beenfound to possess a surprising and unexpected binding affinity for hM₂and hM₃ muscarinic receptor subtypes compared to related-compounds.Additionally, compounds of this invention have been found to havesurprising and unexpected lung selectivity when administered byinhalation thereby resulting in reduced systemic side effects. Moreover,compounds of this invention have been found to possess a surprising andunexpectedly duration of bronchoprotection when administered byinhalation.

Accordingly, in one of its composition aspects, this invention providesa compound of formula I:

wherein

each R¹ and R² is independently selected from C₁₋₄ alkyl, C₂₋₄ alkenyl,C₂₋₄ alkynyl, C₃₋₆ cycloalkyl, cyano, halo, —OR^(a), —SR^(a),—S(O)R^(a), —S(O)₂R^(a) and —NR^(b)R^(c); or two adjacent R¹ groups ortwo adjacent R² groups are joined to form C₃₋₆ alkylene, —(C₂₋₄alkylene)—O— or —O—(C₁₋₄ alkylene)—O—;

each R³ is independently selected from C₁₋₄ alkyl and fluoro;

each R⁴ is independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉ heteroaryl,C₃₋₆ heterocyclic, —CH₂—R⁶ and —CH₂CH₂—R⁷; or both R⁴ groups are joinedtogether with the nitrogen atom to which they are attached to form C₃₋₆heterocyclic;

R⁵ is selected from C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, and —CH₂—R⁸; wherein each alkyl, alkenyl and alkynyl groupis optionally substituted with —OH or 1 to 5 fluoro substituents;

each R⁶ is independently selected from C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl and C₃₋₆ heterocyclic;

each R⁷ is independently selected from C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl, C₃₋₆ heterocyclic, —OH, —O(C₁₋₆ alkyl), —O(C₃₋₆ cycloalkyl),—O(C₆₋₁₀ aryl), —O(C₂₋₉ heteroaryl), —S(C₁₋₆ alkyl), —S(O)(C₁₋₆ alkyl),—S(O)₂(C₁₋₆ alkyl), —S(C₃₋₆ cycloalkyl), —S(O)(C₃₋₆ cycloalkyl),—S(O)₂(C₃₋₆ cycloalkyl), —S(C₆₋₁₀ aryl), —S(O)(C₆₋₁₀ aryl), —S(O)₂(C₆₋₁₀aryl), —S(C₂₋₉ heteroaryl), —S(O)(C₂₋₉ heteroaryl) and —S(O)₂(C₂₋₉heteroaryl);

each R⁸ is independently selected from C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl and C₃₋₆ heterocyclic;

each R^(a) is independently selected from hydrogen, C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl and C₃₋₆ cycloalkyl;

each R^(b) and R^(c) is independently selected from hydrogen, C₁₋₄alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl and C₃₋₆ cycloalkyl; or R^(b) andR^(c) are joined together with the nitrogen atom to which they areattached to form C₃₋₆ heterocyclic;

a is an integer from 0 to 3;

b is an integer from 0 to 3;

c is an integer from 0 to 4;

d is 1 or 2;

e is 8 or 9;

wherein each alkyl, alkylene, alkenyl, alkynyl and cycloalkyl group inR¹, R², R³, R⁴, R⁷, R^(a), R^(b) and R^(c) is optionally substitutedwith 1 to 5 fluoro substituents; each aryl, cycloalkyl, heteroaryl andheterocyclic group in R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R^(a), R^(b) and R^(c)is optionally substituted with 1 to 3 substituents independentlyselected from C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, cyano, halo,—O(C₁₋₄ alkyl), —S(C₁₋₄ alkyl), —S(O)(C₁₋₄ alkyl), —S(O)₂(C₁₋₄ alkyl),—NH₂, —NH(C₁₋₄ alkyl) and —N(C₁₋₄ alkyl)₂, wherein each alkyl, alkylene,alkenyl and alkynyl group is optionally substituted with 1 to 5 fluorosubstituents; and each —CH₂— group in —(CH₂)_(e)— is optionallysubstituted with 1 or 2 substituents independently selected from C₁₋₂alkyl, —OH and fluoro;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

In another of its composition aspects, this invention provides acompound of formula II:

wherein R⁵ and e are as defined herein; or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof.

In separate and distinct embodiments, this invention is also directed tocompounds of formula II wherein the stereochemistry at the 3-position ofthe pyrrolidine ring has the (R) configuration; and compounds of formulaII wherein the stereochemistry at the 3-position of the pyrrolidine ringhas the (S) configuration.

In another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI, or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof. Such pharmaceutical compositions may optionally contain othertherapeutic agents. Accordingly, in one embodiment, this invention isdirected to such a pharmaceutical composition wherein the compositionfurther comprises a therapeutically effective amount of a steroidalanti-inflammatory agent, such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof.

The compounds of this invention are muscarinic receptor antagonists.Accordingly, in one of its method aspects, this invention provides amethod for treating a mammal having a medical condition which isalleviated by treatment with a muscarinic receptor antagonist, themethod comprising administering to the mammal a therapeuticallyeffective amount of a compound of formula I, or apharmaceutically-acceptable salt or solvate or stereoisomer thereof.

In another of its method aspects, this invention provides a method fortreating a pulmonary disorder, the method comprising administering to apatient in need of treatment a therapeutically effective amount of acompound of formula I, or a pharmaceutically-acceptable salt or solvateor stereoisomer thereof.

In yet another of its method aspects, this invention provides a methodof producing bronchodilation in a patient, the method comprisingadministering by inhalation to the patient a bronchodilation-producingamount of a compound of formula I, or a pharmaceutically-acceptable saltor solvate or stereoisomer thereof.

In still another of its method aspects, this invention provides a methodfor treating chronic obstructive pulmonary disease or asthma, the methodcomprising administering to a patient in need of treatment atherapeutically effective amount of a compound of formula I or apharmaceutically-acceptable salt or solvate or stereoisomer thereof.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools for studyingbiological systems or samples having a muscarinic receptor or forstudying the activity of other chemical compounds. Accordingly, in yetanother of its method aspects, this invention provides a method forusing a compound of formula I or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof as a research tool for studying abiological system or sample, or for discovering new chemical compoundshaving muscarinic receptor antagonist activity.

This invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I or a salt or solvate orstereoisomer thereof. Accordingly, in another of its method aspects,this invention provides a process of preparing a compound of formula I,the process comprising:

(a) reacting a compound of formula III with a compound of formula IV inthe presence of a reducing agent;

(b) reacting a compound of formula V with a compound of formula VI inthe presence of a reducing agent;

(c) reacting a compound of formula VII with a compound of formula IV; or

(d) reacting a compound of formula V with a compound of formula VIII;and then

(e) removing any protecting groups to provide a compound of formula I ora salt thereof; wherein the compounds of formulae I and III–VIII are asdefined herein.

In one embodiment, the above process further comprises the additionalstep of forming a pharmaceutically-acceptable salt of a compound offormula I.

In another of its method aspects, this invention provides a process forpreparing a pharmaceutically-acceptable salt of a compound of formula I,the process comprising contacting a compound of formula IX:

wherein R¹–R⁵ and a-e are as defined herein; and P^(a) is an acid-labileamino-protecting group; with a pharmaceutically-acceptable acid to forma pharmaceutically-acceptable salt of a compound of formula I.

In other embodiments, this invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

In another of its composition aspects, this invention provides acompound of formula X:

wherein R¹–R⁵ and a-e are as defined herein; and P is anamino-protecting group; or a salt or solvate or stereoisomer thereof;for use as an intermediate for preparing compounds of formula I.

In another of its composition aspects, this invention provides acompound of formula XI:

wherein R⁵ and e are as defined herein; P is an amino-protecting group;and G is selected from —CHO, —CH(OR^(f))₂, —CH₂OH and —CH₂—L, whereineach R^(f) is independently C₁₋₆ alkyl or both R^(f) groups are joinedto form C₂₋₆ alkylene; and L is a leaving group; or a salt orstereoisomer thereof; for use as an intermediate for preparing compoundsof formula I; provided that when L is chloro, P is not ethoxycarbonyl(i.e., CH₃CH₂OC(O)—).

In additional separate and distinct aspects, this invention provides:

a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for use in therapy;

a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for use as a medicament;

a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for use in treating a pulmonarydisorder, including chronic obstructive pulmonary disease and asthma;

a medicament containing a compound of formula I, or apharmaceutically-acceptable salt or solvate or stereoisomer thereof;

use of a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for treatment of a pulmonary disorder,including chronic obstructive pulmonary disease and asthma;

use of a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, as a medicament for the treatment of apulmonary disorder, including chronic obstructive pulmonary disease andasthma;

use of a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for manufacture of a medicament; and

use of a compound of formula I, or a pharmaceutically-acceptable salt orsolvate or stereoisomer thereof, for manufacture of a medicament fortreatment of a pulmonary disorder, including chronic obstructivepulmonary disease and asthma.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel substituted pyrrolidine and relatedcompounds of formula I, or pharmaceutically-acceptable salts or solvatesor stereoisomers thereof. These compounds may contain one or more chiralcenters and, when such a chiral center or centers are present, thisinvention is directed to racemic mixtures; pure stereoisomers (i.e.,individual enantiomers or diastereomers); and stereoisomer-enrichedmixtures of such isomers, unless otherwise indicated. When a particularstereoisomer is shown, it will be understood by those skilled in the artthat minor amounts of other stereoisomers may be present in thecompositions of this invention unless otherwise indicated, provided thatthe utility of the composition as a whole is not eliminated by thepresence of such other isomers.

The compounds of this invention also contain several basic groups (e.g.,amino groups) and therefore, the compounds of formula I can exist as thefree base or in various salt forms. All such forms are included withinthe scope of this invention. Also included within the scope of thisinvention are pharmaceutically-acceptable solvates of the compounds offormula I or the salts thereof.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds offormula I are included within the scope of this invention unlessotherwise specified.

The nomenclature used herein to name the compounds of this invention isillustrated in the Examples herein. Generally, this nomenclature hasbeen derived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.).

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples and embodiments of various aspects of thisinvention. These representative values are intended to further definesuch aspects and embodiments and are not intended to exclude otherembodiments or limit the scope of this invention. In this regard, therepresentation herein that a particular value or substituent ispreferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

In a specific embodiment, R¹ or R², when present, are independentlyselected from C₁₋₄ alkyl, fluoro, chloro and —OR^(a); wherein each alkylgroup is optionally substituted with 1 to 3 fluoro substituents. Inanother specific embodiment, each R¹ and R² is C₁₋₂ alkyl or fluoro.Representative R¹ and R² groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, difluoromethyl, trifluoromethyl,2,2,2-trifluoroethyl, fluoro, chloro, methoxy, ethoxy, difluoromethoxyand trifluoromethoxy.

In a specific embodiment, each R³, when present, is independentlyselected from C₁₋₂ alkyl and fluoro; wherein each alkyl group isoptionally substituted with 1 to 3 fluoro substituent. When two R³substituents are present, they can be on the same or different carbonatoms. Representative R³ groups include, but are not limited to, methyl,ethyl, difluoromethyl, trifluoromethyl and fluoro.

In specific embodiments, each R⁴ is independently hydrogen or C₁₋₄alkyl; or each R⁴ is independently hydrogen or C₁₋₂ alkyl; or each R⁴ ishydrogen. Representative R⁴ groups include, but are not limited to,hydrogen, methyl and ethyl.

Alternatively, in another specific embodiment, both R⁴ groups are joinedtogether with the nitrogen atom to which they are attached to form aC₃₋₅ heterocyclic ring optionally containing one additional heteroatomselected from nitrogen, oxygen or sulfur. Representative heterocyclicrings include, but are not limited to, pyrrolidin-1-yl, piperidin-1-yl,piperazin-1-yl, morpholin-4-yl and thiomorpholin-4-yl.

In specific embodiments, R⁵ is C₁₋₅ alkyl; or R⁵ is C₁₋₄ alkyl; or R⁵ isC₁₋₃ alkyl; or R⁵ is C₁₋₂ alkyl; wherein the alkyl group is optionallysubstituted with —OH or 1 to 3 fluoro substituents. Representative R⁵groups in this embodiment include, but are not limited to, methyl,ethyl, 2-hydroxyethyl, 2,2,2-trifluoroethyl, n-propyl, isopropyl,1-hydroxyprop-2-yl, n-butyl and isobutyl. In one embodiment, R⁵ ismethyl.

In other specific embodiments, R⁵ is C₃₋₅ cycloalkyl; or R⁵ is C₃₋₄cycloalkyl; wherein the cycloalkyl group is optionally substituted with—OH or 1 to 3 fluoro substituents. Representative R⁵ groups in thisembodiment include, but are not limited to, cyclopropyl, cyclobutyl andcyclopentyl.

In another specific embodiment, R⁵ is —CH₂—R⁸, wherein R⁸ is as definedherein. In separate aspects of this embodiment, R⁵ (i.e., —CH₂—R⁸) isselected from:

(a) —CH₂—(C₃₋₅ cycloalkyl); or —CH₂—(C₃₋₄ cycloalkyl); wherein thecycloalkyl group is optionally substituted with —OH or 1 to 3 fluorosubstituents;

(b) —CH₂-(phenyl), i.e., benzyl, wherein the phenyl group is optionallysubstituted with 1 to 3 substituents independently selected from C₁₋₄alkyl, cyano, fluoro, chloro, —O(C₁₋₄ alkyl), —S(C₁₋₄ alkyl) and—S(O)₂(C₁₋₄ alkyl); where each alkyl group is optionally substitutedwith 1 to 3 fluoro substituent.

Representative R⁵ groups in this embodiment include, but are not limitedto, cyclopropylmethyl, cyclobutylmethyl and cyclopentylmethyl; andbenzyl, 4-cyanobenzyl, 3-methylbenzyl, 4-methylbenzyl,4-trifluoromethoxybenzyl, 3-fluorobenzyl and 4-fluorobenzyl.

In a specific embodiment, each R⁶ is independently phenyl; wherein eachphenyl group is optionally substituted with 1 to 3 substituentsindependently selected from C₁₋₄ alkyl, cyano, fluoro, chloro, —O(C₁₋₄alkyl), —S(C₁₋₄ alkyl) and —S(O)₂(C₁₋₄ alkyl); where each alkyl group isoptionally substituted with 1 to 3 fluoro substituent.

In a specific embodiment, each R⁷ is independently selected from phenyl,—OH and —O(C₁₋₂ alkyl); wherein each alkyl group is optionallysubstituted with 1 to 3 fluoro substituent; and each phenyl group isoptionally substituted with 1 to 3 substituents independently selectedfrom C₁₋₄ alkyl, cyano, fluoro, chloro, —O(C₁₋₄ alkyl), —S(C₁₋₄ alkyl)and —S(O)₂(C₁₋₄ alkyl); where each alkyl group is optionally substitutedwith 1 to 3 fluoro substituent.

In specific embodiments, each R^(a) is independently selected fromhydrogen and C₁₋₃ alkyl; or hydrogen and C₁₋₂ alkyl; wherein each alkylgroup is optionally substituted with 1 to 3 fluoro substituent.Representative R^(a) groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, difluoromethyl, trifluoromethyl and2,2,2-trifluoroethyl.

In specific embodiments, each R^(b) and R^(c) is independently selectedfrom hydrogen and C₁₋₃ alkyl; or hydrogen and C₁₋₂ alkyl; wherein eachalkyl group is optionally substituted with 1 to 3 fluoro substituent.Representative R^(b) and R^(c) groups include, but are not limited to,methyl, ethyl, n-propyl, isopropyl, difluoromethyl, trifluoromethyl and2,2,2-trifluoroethyl.

Alternatively, in another specific embodiment, R^(b) and R^(c) arejoined together with the nitrogen atom to which they are attached toform a C₃₋₅ heterocyclic ring optionally containing one additionalheteroatom selected from nitrogen, oxygen or sulfur. Representativeheterocyclic rings include, but are not limited to, pyrrolidin-1-yl,piperidin-1-yl, piperazin-1-yl, morpholin-4-yl and thiomorpholin-4-yl.

In specific embodiments, a is 0, 1 or 2; or a is 0 or 1; or a is 0.

In specific embodiments, b is 0, 1 or 2; or b is 0 or 1; or b is 0.

In specific embodiments, c is 0, 1 or 2; or c is 0 or 1; or c is 0.

When d is 1, i.e., when the ring defined by d is a pyrrolidine ring,then in one embodiment, the stereocenter at the 3-position of thepyrrolidine ring (i.e., the carbon atom bearing the1-carbamoyl-1,1-diphenylmethyl group) has the (S) stereochemistry. Inanother embodiment, this stereocenter has the (R) stereochemistry.

In one embodiment, e is 8. In another embodiment, e is 9.

A particular embodiment of the present invention are compounds offormula I wherein both R⁴ groups are hydrogen, a, b and c are 0; d is 1;e is 8 or 9; and R⁵ is C₁₋₃ alkyl; or C₁₋₂ alkyl; or apharmaceutically-acceptable salt or solvate or stereoisomer thereof.

Another particular embodiment of the present invention are compounds offormula I wherein both R⁴ groups are hydrogen, a, b and c are 0; d is 1;e is 8 or 9; and R⁵ is C₃₋₅ cycloalkyl; or C₃₋₄ cycloalkyl; or apharmaceutically-acceptable salt or solvate or stereoisomer thereof.

Yet another particular embodiment of the present invention are compoundsof formula I wherein R⁵ is methyl; and R¹, R², R³, R⁴, a, b, c, d, and eare as defined herein; or a pharmaceutically-acceptable salt or solvateor stereoisomer thereof.

Other specific embodiments of the present invention are compounds offormula IIa:

wherein R⁵ and e are as defined in Table I, or apharmaceutically-acceptable salt or solvate thereof.

TABLE I Example No. R⁵ e 1 —CH₃ 8 2 —CH(CH₃)₂ 8 3 —CH₂CH₂CH₃ 8 4-cyclopropyl 8 5 -cyclobutyl 8 6 -cyclopentyl 8 7 —CH₂CH₃ 8 8 —CH₂CH₂OH8 9 —CH(CH₃)CH₂OH (R)-isomer 8 10 —CH(CH₃)CH₂OH 8 11 —CH(CH₃)CH₂OH(S)-isomer 8 12 —CH₂CF₃ 8 13 —CH₂Ph^(†) 8 14 —CH₃ 9 15 —CH(CH₃)₂ 9 16—CH₂CH₂CH₃ 9 17 -cyclopropyl 9 18 -cyclobutyl 9 19 -cyclopentyl 9 20—CH₂CH₃ 9 21 —CH₂CH₂OH 9 22 —CH(CH₃)CH₂OH (R)-isomer 9 23 —CH(CH₃)CH₂OH9 24 —CH(CH₃)CH₂OH (S)-isomer 9 25 —CH₂CF₃ 9 26 —CH₂Ph 9 ^(†)Ph =phenyl.

Still other specific embodiments of the present invention are compoundsof formula IIb:

wherein R⁵ and e are as defined in Table II, or apharmaceutically-acceptable salt or solvate thereof.

TABLE II Example No. R⁵ e 27 —CH₃ 8 28 —CH(CH₃)₂ 8 29 —CH₂CH₂CH₃ 8 30-cyclopropyl 8 31 -cyclobutyl 8 32 -cyclopentyl 8 33 —CH₂CH₃ 8 34—CH₂CH₂OH 8 35 —CH(CH₃)CH₂OH (R)-isomer 8 36 —CH(CH₃)CH₂OH 8 37—CH(CH₃)CH₂OH (S)-isomer 8 38 —CH₂CF₃ 8 39 —CH₂Ph^(†) 8 40 —CH₃ 9 41—CH(CH₃)₂ 9 42 —CH₂CH₂CH₃ 9 43 -cyclopropyl 9 44 -cyclobutyl 9 45-cyclopentyl 9 46 —CH₂CH₃ 9 47 —CH₂CH₂OH 9 48 —CH(CH₃)CH₂OH (R)-isomer 949 —CH(CH₃)CH₂OH 9 50 —CH(CH₃)CH₂OH (S)-isomer 9 51 —CH₂CF₃ 9 52 —CH₂Ph9 ^(†)Ph = phenyl.

Still other specific embodiments of the present invention are compoundsof formula XII:

wherein R⁵ and e are as defined in Table III, or apharmaceutically-acceptable salt or solvate thereof.

TABLE III Example No. R⁵ e 53 —CH₃ 8 54 —CH(CH₃)₂ 8 55 —CH₂CH₂CH₃ 8 56-cyclopropyl 8 57 -cyclobutyl 8 58 -cyclopentyl 8 59 —CH₂CH₃ 8 60—CH₂CH₂OH 8 61 —CH(CH₃)CH₂OH (R)-isomer 8 62 —CH(CH₃)CH₂OH 8 63—CH(CH₃)CH₂OH (S)-isomer 8 64 —CH₂CF₃ 8 65 —CH₂Ph^(†) 8 66 —CH₃ 9 67—CH(CH₃)₂ 9 68 —CH₂CH₂CH₃ 9 69 -cyclopropyl 9 70 -cyclobutyl 9 71-cyclopentyl 9 72 —CH₂CH₃ 9 73 —CH₂CH₂OH 9 74 —CH(CH₃)CH₂OH (R)-isomer 975 —CH(CH₃)CH₂OH 9 76 —CH(CH₃)CH₂OH (S)-isomer 9 77 —CH₂CF₃ 9 78 —CH₂Ph9 ^(†)Ph = phenyl.

In the compounds of formulas X and XI, P is an amino-protecting group.In one embodiment, P is an acid labile amino-protecting group (P^(a)).In another embodiment, P is selected from benzyl, 4-methoxybenzyl,2,4-dimethoxybenzyl, diphenylmethyl, triphenylmethyl, methoxycarbonyl,ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl,p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, acetyl,trimethylsilyl and tert-butyldimethylsilyl. In a particular embodiment,P is tert-butoxycarbonyl.

In the compounds of formula XI, L is a leaving group. In one embodiment,L is chloro, bromo or iodo. In another embodiment, L ismethanesulfonyloxy(mesylate) or p-toluenesulfonyloxy(tosylate). In aparticular embodiment, L is p-toluenesulfonyloxy.

In one embodiment, R^(f) is methyl or ethyl. In another embodiment, bothR^(f) groups are joined to form —(CH₂)₂— or —(CH₂)₃—.

Particular compounds of formula X of interest are:

2-[(S)-1-(8-N-Benzyl-N-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;and

2-{(S)-1-[8-(N-tert-Butoxycarbonyl-N-methylamino)octy]pyrrolidin-3-yl}-2,2-diphenylacetamide.

Particular compounds of formula XI of interest are:

8-(N-benzyl-N-methylamino)octan-1-ol;

8-(N-tert-butoxycarbonyl-N-methylamino)octan-1-ol; and

toluene-4-sulfonic acid 8-(N-tert-butoxycarbonyl-N-methylamino)octylester.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene,ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl,butane-1,4-diyl, pentane-1,5-diyl and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom.

The term “pharmaceutically-acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically-acceptable inorganic ororganic bases and from pharmaceutically-acceptable inorganic or organicacids. Salts derived from pharmaceutically-acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularsalts of interest are ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically-acceptable organic basesinclude salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically-acceptable acidsinclude acetic, ascorbic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, edisylic, fumaric, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, xinafoicand the like. Particular salts of interest are citric, hydrobromic,hydrochloric, isethionic, maleic, phosphoric, sulfuric and tartaricacids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Preferably, the salt is apharmaceutically-acceptable salt, although this is not required forsalts of intermediate compounds which are not intended foradministration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. This term alsoincludes clathrates, including clathrates with water. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “bronchoprotection” or “bronchoprotective” means preventing,ameliorating, suppressing or alleviating the symptoms of a respiratorydisease or disorder. For purposes of determining the duration ofbronchoprotection, the guinea pig model of acetylcholine-inducedbronchoconstriction is used unless otherwise indicated.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD or asthma) ina patient, such as a mammal (particularly a human or a companion animal)which includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to, benzyl,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),p-methoxybenzyloxycarbonyl (Moz), 9-fluorenylmethoxycarbonyl (Fmoc),formyl, acetyl, trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), andthe like. The term “acid labile amino-protecting group” means anamino-protecting group that is removed by treatment with an acidincluding, for example, a mineral acid or an organic acid, such as acarboxylic acid or a sulfonic acid. Representative acid labileamino-protecting groups include, but are not limited to, carbamates suchas tert-butoxycarbonyl (BOC), p-methoxybenzyloxycarbonyl (Moz) and thelike.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluroenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “optionally substituted” means that the specified group ormoiety, such as an alkyl group, phenyl group and the like, isunsubstituted or is substituted with the specified substituents.

General Synthetic Procedures

The substituted pyrrolidine and related compounds of this invention canbe prepared from readily available starting materials using thefollowing general methods and procedures. Although a particularembodiment of the present invention may be shown or described in theSchemes below, those skilled in the art will recognize that allembodiments or aspects of the present invention can be prepared usingthe methods described herein or by using other methods, reagents andstarting materials known to those skilled in the art. It will also beappreciated that where typical or preferred process conditions (i.e.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. The optimum reaction conditions may vary withthe particular reactants or solvent used, but such conditions can bereadily determined by one skilled in the art by routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

The compounds of formula I or salts thereof can be prepared by a processcomprising:

(a) reacting a compound of formula III:

with a compound of formula IV:

wherein P¹ is an amino-protecting group, in the presence of a reducingagent;

(b) reacting a compound of formula V:

with a compound of formula VI:

wherein P² is an amino-protecting group, in the presence of a reducingagent;

(c) reacting a compound of formula VII:

wherein L¹ is a leaving group, with a compound of formula IV; or

(d) reacting a compound of formula V with a compound of formula VIII:

wherein L² is a leaving group and P³ is an amino-protecting group; andthen

(e) removing protecting group P¹, P² or P³ to provide a compound offormula I or a salt thereof; wherein R¹⁻⁵ and a-e are as defined herein.

Optionally, a pharmaceutically-acceptable salt of the compound offormula I can be prepared directly in step (e) or, as a separateadditional step, from the product of step (e).

In process (a), P¹ can be any suitable amino-protecting group, such asbenzyl and the like. The reducing agent can be any suitable reducingagent, including metal hydride reducing agents, such as sodiumtriacetoxyborohydride, sodium cyanoborohydride and the like. Uponcompletion of the reaction, the amino-protecting group, P¹, can beremoved using conventional procedures and reagents. For example, abenzyl protecting group can be removed by hydrogenolysis in the presenceof a catalyst, such as pallidum.

In process (b), P² can be any suitable amino-protecting group, such asbenzyl, tert-butoxycarbonyl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, tert-butyldimethylsilyl and the like. Thereducing agent can be any suitable reducing agent, including metalhydride reducing agents, such as sodium triacetoxyborohydride, sodiumcyanoborohydride and the like. Upon completion of the reaction, theamino-protecting group, P², can be removed using conventional proceduresand reagents. For example, a benzyl protecting group can be removed byhydrogenolysis in the presence of a catalyst, such as pallidum; atert-butoxycarbonyl group can be removed by treatment with acid, such ashydrochloric acid, p-toluenesulfonic acid and the like; atert-butyldimethylsilyl group can be removed by treatment with a sourceof fluoride ions, such as triethylamine trihydrofluoride.

In process (c), L¹ can be any suitable leaving group including, but notlimited to halo, such as chloro, bromo or iodo, or a sulfonic estergroup, such as mesylate, tosylate and the like; and P¹ is as definedherein.

In process (d), L² can be any suitable leaving group including, but notlimited to, halo, such as chloro, bromo or iodo, or a sulfonic estergroup, such as mesylate, tosylate and the like; and P³ can be anysuitable amino-protecting group, such as benzyl, tert-butoxycarbonyl,benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, tert-butyldimethylsilyland the like. The reducing agent can be any suitable reducing agent,including metal hydride reducing agents, such as sodiumtriacetoxyborohydride, sodium cyanoborohydride and the like. Uponcompletion of the reaction, the amino-protecting group, P², can beremoved using conventional procedures and reagents. For example, abenzyl protecting group can be removed by hydrogenolysis in the presenceof a catalyst, such as pallidum; a tert-butoxycarbonyl group can beremoved by treatment with acid, such as hydrochloric acid,p-toluenesulfonic acid and the like; a tert-butyldimethylsilyl group canbe removed by treatment with a source of fluoride ions, such astriethylamine trihydrofluoride.

In particular embodiments of processes (b) and (d), P² and P³ are atert-butoxycarbonyl group which is removed by treatment with apharmaceutically-acceptable acid to generate in situ apharmaceutically-acceptable salt of the compound of formula I.

By way of further illustration, the preparation of representativecompounds of formula I is shown in Scheme A (where the substituents andvariables shown in the following Schemes have the definitions providedherein unless otherwise indicated).

As shown in Scheme A, a compound of formula 1 is first reacted withalcohol 2, where L³ is a suitable leaving group, such as chloro, bromo,iodo, tosyl, mesyl and the like, to provide intermediate 3. Typically,this reaction is conducted by contacting 1 with at least one equivalent,preferably with about 1.0 to about 1.1 equivalents, of alcohol 2 in aninert diluent, such as acetonitrile and the like. This reaction isgenerally conducted in presence of excess base; preferably, in thepresence of about 2 to about 4 equivalent of a base, such as atrialkylamine, preferably triethylamine. Typically, this reaction isconducted at a temperature ranging from about 0° C. to about 80° C.,preferably about 40° C. to 50° C., for about 1 to 24 hours, or until thereaction is substantially complete. If desired, the resultingintermediate 3 is readily purified by standard procedures, such aschromatography, recrystallization and the like.

The alcohols of formula 2 used in this reaction are either commerciallyavailable or can be prepared from commercially available startingmaterials and reagents using well-known procedures. Representativealcohols of formula 2 include, by way of example, 8-chloro-1-octanol,9-chloro-1-nonanol, 8-bromo-1-octanol, 9-bromo-1-nonanol,8-iodo-1-octanol, 9-iodo-1-nonanol and the like.

The hydroxyl group of intermediate 3 is then oxidized to thecorresponding aldehyde to provide intermediate 4. This reaction istypically conducted by contacting 3 with an excess amount of a suitableoxidizing agent. Any oxidizing agent capable of oxidizing a hydroxylgroup to an aldehyde may be used in this reaction including chromium(VI) reagents, such as dipyridine chromium (VI) oxide, pyridiniumchlorochromate, pyridinium dichromate and the like; and activateddimethyl sulfoxide reagents, such oxalyl chloride/DMSO, sulfur trioxidepyridine complex/DMSO/trialkylamine and the like.

Preferably, this reaction is conducted using an excess of sulfurtrioxide pyridine complex and dimethyl sulfoxide in the presence of atrialkylamine, such as triethylamine, diisopropylethylamine and thelike. Typically, this reaction is conducted by contacting 3 with about2.5 to about 3.5 equivalents of sulfur trioxide pyridine complex and anexcess, preferably about 10 equivalents, of dimethyl sulfoxide in thepresence of an excess, preferably about 5 equivalents, ofdiisopropylethylamine in an inert diluent, such as dichloromethane. Thisreaction is generally conducted at a temperature ranging from about −30°C. to about 0° C., preferably at about −10° C. to about −20° C., forabout 0.25 to about 2 hours, or until the reaction is substantiallycomplete. Optionally, the resulting aldehyde intermediate 4 is thenpurified using standard procedures, such as chromatography,recrystallization and the like.

Alternatively, aldehyde intermediate 4 can be prepared by first reacting1 with a compound of the formula:

wherein L⁴ and L⁵ are suitable leaving groups, such as chloro, bromo,iodo, tosyl, mesyl and the like, e is as defined herein, and each R^(d)is independently C₁₋₆ alkyl or both R^(d) groups are joined to form C₂₋₆alkylene. Subsequent, hydrolysis of the acetal (i.e., using aqueousacid) or ozonolysis of the olefin (i.e., using O₃, followed bydecomposition of the ozonide with a reducing agent, such as trimethylphosphite, dimethyl sulfide and the like) then affords aldehyde 4.

Aldehyde intermediate 4 is then coupled with amine 5 to afford acompound of formula 6. Typically, this reaction is conducted bycontacting aldehyde 4 with an excess, such as about 1.0 to about 1.2equivalents, of 5 in the presence of an excess, preferably about 1.2 toabout 1.5 equivalent, of a suitable reducing agent in an inert diluent,such as dichloromethane. Suitable reducing agents include, by way ofillustration, sodium triacetoxyborohydride, sodium cyanoborohydride andthe like. Preferably, the reducing agent is sodiumtriacetoxyborohydride. Generally, this reaction is conducted at atemperature ranging from about 0° C. to about 30° C. for about 6 toabout 24 hours, or until the reaction is substantially complete. Theresulting compound of formula 6 is typically purified using standardprocedures, such as chromatography, recrystallization and the like.

Removal of the benzyl group from 6 using conventional reagents andreaction conditions then affords 7. For example, hydrogenolysis of 6using a catalyst, such as palladium on carbon and/or palladiumhydroxide, readily removes the benzyl group to provide 7. Typically,this reaction is conducted by contacting 6 with hydrogen at a pressureranging from about 40 to about 60 psi in the presence of a catalyst,such as 10% palladium on carbon. This reaction is generally conducted inan inert diluent, such as ethanol or isopropanol, at ambient temperaturefor about 12 to 120 hours, or until the reaction is substantiallycomplete.

Alternatively, aldehyde intermediate 5 can be reacted with an amine ofthe formula R⁵—NH₂, where R⁵ is as defined herein, to afford compound 7directly. Additionally, if desired, other amino-protecting groups may beused in place of the benzyl group in Scheme A.

The amine compounds suitable for use in the reactions described hereinare either commercially available or can be prepared from commerciallyavailable starting materials and reagents using well-known procedures.Representative amines suitable for use include, but are not limited to,N-methyl-N-benzylamine, N-ethyl-N-benzylamine, methylamine, ethylamine,n-propylamine, isopropylamine, 2-hydroxyethylamine,DL-2-amino-1-propanol, (R)-(−)-2-amino-1-propanol,(S)-(+)-2-amino-1-propanol, 2,2,2-trifluoroethylamine, benzylamine,cyclopropylamine, cyclobutylamine, cyclopentylamine and the like.

The compounds of formula 1 employed in the reactions described hereinare readily prepared by the procedures illustrated in Scheme B.

As illustrated in Scheme B, diphenylacetonitrile 8 is reacted withintermediate 9, where L⁶ is a suitable leaving group, such as chloro,bromo, iodo, tosyl, mesyl and the like, and P⁴ is an amino-protectinggroup, such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, ethoxycarbonyl,phenylcarbonyl and the like, to provide intermediate 10. Typically, thisreaction is conducted by first forming the anion of compound 8 bycontacting 8 with excess, preferably about 1.4 to about 1.6 equivalents,of a strong base, such as potassium tert-butoxide, in an inert diluent,such as tetrahydrofuran, at a temperature ranging from about −10° C. toabout 10° C. for about 0.5 to about 2.0 hours. The resulting anion isthen reacted in situ with about 0.95 to about 1.05 equivalents of 9 at atemperature ranging from about 20° C. to about 50° C. for about 10 toabout 48 hours, or until the reaction is substantially complete.Compounds of formula 9, where L⁶ is a sulfonate ester leaving group, arereadily prepared from the corresponding alcohol using conventionalprocedures and reagents. For example, (S)-1-benzyl-3-pyrrolidinol isreadily converted to (S)-1-benzyl-3-(p-toluenesulfonyloxy)pyrrolidine bytreatment with about 1.1 equivalents of p-toluenesulfonyl chloride andabout 1.2 equivalents of 1,4-diazabicyclo[2.2.2]octane (DABCO). Othercompounds of formula 9 can be prepared by similar procedures usingcommercially available starting materials and reagents.

Compound 10 is then deprotected using conventional procedures andreagents to afford compound 11. For example, if P⁴ in compound 10 is abenzyl protecting group, the benzyl group is readily removed by transferhydrogenolysis using a hydrogen source, such as ammonium formate, and acatalyst, such as palladium on carbon. Preferably, this reaction isconducted using the hydrochloride or hydrobromide salt of compound 10 orin the presence of an acid, such as hydrochloric acid, hydrobromic acid,formic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid,acetic acid, oxalic acid and the like. This hydrogenolysis reaction canalso be conducted using hydrogen and a catalyst in the presence of anacid. See, for example, U.S. Pat. No. 6,005,119, issued Dec. 21, 1999 toN. Mori et al.

The nitrile group of compound 11 is then hydrolyzed to the correspondingamide (i.e., —C(O)NH₂) to provide a compound of formula 10. Thisreaction is typically conducted by contacting 11 with aqueous sulfuricacid, preferably 80% sulfuric acid, at a temperature ranging from about70° C. to about 100° C., preferably about 90° C., for about 12 to about36 hours, or until the reaction is substantially complete. As shown inScheme B, hydrolysis of the nitrile group to the amide can also beperformed before removal of the protecting group to afford 13, which canthen be deprotected to provide compound 12.

If desired, the nitrile group of compound 10 or 11 can be hydrolyzed tothe corresponding carboxylic acid (i.e., —COOH) using, for example,aqueous sodium hydroxide containing about 6 to about 12% hydrogenperoxide. The resulting carboxylic acid can then be coupled to variousamines (i.e., R^(e)R^(e)NH, where R^(e) is as defined herein) to formsubstituted amides using well-known procedures and reagents.

Compounds of this invention can also be prepared by the procedureillustrated in Scheme C.

As shown in Scheme C, alcohol 2, where L⁷ is a suitable leaving group,such as chloro, bromo, iodo, tosyl, mesyl and the like, can be reactedwith benzylamine 5 to provide intermediate 14. Typically, this reactionis conducted by contacting alcohol 2 with at least one equivalent,preferably with about 1.0 to about 1.1 equivalents, of benzylamine 5 inan inert diluent, such as acetonitrile and the like. This reaction isgenerally conducted in presence of excess base; preferably, in thepresence of about 2 to about 4 equivalent of a base, such as atrialkylamine, preferably triethylamine. Typically, this reaction isconducted at a temperature ranging from about 0° C. to about 80° C.,preferably about 40° C. to 60° C., for about 1 to 24 hours, or until thereaction is substantially complete. If desired, the resultingintermediate 14 is readily purified by standard procedures, such aschromatography, recrystallization and the like.

The hydroxyl group of intermediate 14 is then oxidized to thecorresponding aldehyde to provide intermediate 15. This reaction istypically conducted by contacting 14 with an excess amount of a suitableoxidizing agent. Any oxidizing agent capable of oxidizing a hydroxylgroup to an aldehyde may be used in this reaction including chromium(VI) reagents, such as dipyridine chromium (VI) oxide, pyridiniumchlorochromate, pyridinium dichromate and the like; and activateddimethyl sulfoxide reagents, such oxalyl chloride/DMSO, sulfur trioxidepyridine complex/DMSO/trialkylamine and the like.

Preferably, this reaction is conducted using an excess of sulfurtrioxide pyridine complex and dimethyl sulfoxide in the presence of atrialkylamine, such as triethylamine, diisopropylethylamine and thelike. Typically, this reaction is conducted by contacting 14 with about2.5 to about 3.5 equivalents of sulfur trioxide pyridine complex and anexcess, preferably about 10 equivalents, of dimethyl sulfoxide in thepresence of an excess, preferably about 5 equivalents, ofdiisopropylethylamine in an inert diluent, such as dichloromethane. Thisreaction is generally conducted at a temperature ranging from about −30°C. to about 0° C., preferably at about −10° C. to about −20° C., forabout 0.25 to about 6 hours, or until the reaction is substantiallycomplete. Optionally, the resulting aldehyde intermediate 15 is thenpurified using standard procedures, such as chromatography,recrystallization and the like.

Aldehyde intermediate 15 is then coupled with 1 to afford a compound offormula 6. Typically, this reaction is conducted by contacting aldehyde15 with at least about one equivalent of 1 in the presence of an excess,preferably about 1.2 to about 1.5 equivalent, of a suitable reducingagent in an inert diluent, such as dichloromethane. Suitable reducingagents include, by way of illustration, sodium triacetoxyborohydride,sodium cyanoborohydride and the like. Preferably, the reducing agent issodium triacetoxyborohydride. Generally, this reaction is conducted at atemperature ranging from about 0° C. to about 30° C. for about 2 toabout 24 hours, or until the reaction is substantially complete. Theresulting compound of formula 6 is typically purified using standardprocedures, such as chromatography, recrystallization and the like. Thebenzyl group can then be removed from 6 to afford 7 as discussed above.

Additionally, it will also be appreciated by those skilled in the artthat the synthetic steps illustrated in Schemes A, B and C can beconducted in a different order from that shown, or by using differentreagents from those described, to produce the compounds of formula 7.For example, instead of oxidizing the hydroxyl group of intermediate 3or 14 to an aldehyde, these hydroxyl groups can be converted into aleaving group, such as a chloro, bromo, iodo, mesylate or tosylate,using conventional reagents and reaction procedures. The resultingleaving group is then readily displaced with amine 5 or intermediate 1to afford compound 6.

By way of further example, representative compounds of formula I can beprepared as illustrated in Scheme D:

As shown in Scheme D, the benzyl amino-protecting group of compound 14can be removed and replaced with a tert-butoxycarbonyl amino-protectinggroup using conventional procedures and reagents (i.e., hydrogenolysisto remove the benzyl group and di-tert-butyl dicarbonate to form thetert-butoxycarbonyl group) to provide compound 16.

The hydroxyl group of compound 16 is then converted into a leavinggroup, such as a chloro, bromo, iodo, mesylate or tosylate, usingconventional reagents and reaction procedures to provide a compound offormula 17. For example, the hydroxyl group is converted to a tosylateleaving group by reaction with tosyl chloride (p-toluenesulfonylchloride) in the presence of a suitable base including tertiary amines,such as 1,4-diazabicyclo[2.2.2]octane. This reaction is typicallyconducted in an inert diluent, such as methyl tert-butyl ether, at atemperature ranging from about 0° C. to about 30° C. for 0.5 to 6 hours,or until the reaction is substantially complete.

The leaving group of compound 17 is then displaced with a compound offormula 1 to provide a compound of formula 18. This reaction istypically conducted by contacting 17 with about 0.95 to about 1.1 molarequivalents of 1 in the presence of a tertiary amine, such asdiisopropylethylamine. The reaction is generally conducted in an inertdiluent, such as acetonitrile, at a temperature ranging from about 25°C. to about 100° C. for about 2 to about 12 hours, or until the reactionis substantially complete.

The tert-butoxycarbonyl amino-protecting group of compound 18 is thenremoved using conventional reagents and reaction conditions to afford acompound of formula 7 or a salt thereof. For example, thetert-butoxycarbonyl amino-protecting group can be readily removed bytreatment with an acid, such as hydrochloric acid, trifluoroacetic acid,p-toluenesulfonic acid and the like.

In one embodiment, the compound of formula 18 is contacted with apharmaceutically-acceptable acid to generate apharmaceutically-acceptable salt of compound 7 directly withoutisolation of the free-base. For example, 18 can be contacted withnaphthalene-1,5-disulfonic acid to form the naphthalene-1,5-disulfonicacid salt of compound 7. This reaction is typically conducted bycontacting 18 with about 1 to about 3 equivalents, such as 2equivalents, of naphthalene-1,5-disulfonic acid in an inert diluent,such as isopropanol. In one embodiment, isopropanol containing about 2to about 10% by volume water is employed as the diluent to provide acrystalline naphthalene-1,5-disulfonic acid salt.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereto are described in the Examples set forth below.

Pharmaceutical Compositions

The substituted pyrrolidine and related compounds of this invention aretypically administered to a patient in the form of a pharmaceuticalcomposition. Such pharmaceutical compositions may be administered to thepatient by any acceptable route of administration including, but notlimited to, oral, inhaled, nasal, topical (including transdermal) andparenteral modes of administration.

It will be understood that any form of the compounds of this invention,(i.e., free base, pharmaceutically-acceptable salt, or solvate) that issuitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one of its compositions aspects, this invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula I or II, or a pharmaceuticallyacceptable salt thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically-acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.01 to about 95% byweight of the active agent; including, from about 0.01 to about 30% byweight; such as from about 0.01 to about 10% by weight of the activeagent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially-available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby throughly and intimately mixing or blending a compound of theinvention with a pharmaceutically-acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are providedcommercially, for example by PARI GmbH (Starnberg, German). Othernebulizer devices are disclosed, for example, in U.S. Pat. No. 6,123,068and WO 97/12687.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous saline solution comprising fromabout 0.05 μg/mL to about 10 mg/mL of a compound of formula I or apharmaceutically-acceptable salt or solvate or stereoisomer thereof. Inone embodiment, the pH of this composition is in the range of from about4 to about 6. In a particular embodiment, this composition is optionallybuffered using citrate buffer to a pH of about 5.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose or starch.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry lactose having a particle size between about 1 μmand about 100 μm and micronized particles of a compound of formula I, ora pharmaceutically-acceptable salt or solvate or stereoisomer thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No.6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S.Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S.Pat. No. 4,353,365). Further examples of suitable DPI devices aredescribed in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 andreferences cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or apharmaceutically-acceptable salt thereof using compressed propellantgas. Accordingly, pharmaceutical compositions administered using ametered-dose inhaler typically comprise a solution or suspension of theactive agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including chlorofluorocarbons, such as CCl₃F,and hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3,-heptafluoro-n-propane, (HFA 227). Due toconcerns about chlorofluorocarbons affecting the ozone layer,formulations containing HFAs are generally preferred. Additionaloptional components of HFA formulations include co-solvents, such asethanol or pentane, and surfactants, such as sorbitan trioleate, oleicacid, lecithin, and glycerin. See, for example, U.S. Pat. No. 5,225,183,EP 0717987 A2, and WO 92/22286.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula I, or a pharmaceutically-acceptable salt or solvate orstereoisomer thereof; from about 0% to about 20% by weight ethanol; andfrom about 0% to about 5% by weight surfactant; with the remainder beingan HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of an metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in U.S. Pat. Nos. 6,006,745 and6,143,277. Alternatively, a suspension formulation can be prepared byspray drying a coating of surfactant on micronized particles of theactive agent. See, for example, WO 99/53901 and WO 00/61108.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO99/55319 and WO 00/30614.

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically-acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetra acetic acid (EDDA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Theterm “unit dosage form” means a physically discrete unit suitable fordosing a patient, i.e., each unit containing a predetermined quantity ofactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycolm monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a compound of formulaI, or pharmaceutically-acceptable salt or solvate or stereoisomerthereof. For example, the pharmaceutical compositions of this inventionmay further comprise one or more therapeutic agents selected from β₂adrenergic receptor agonists, anti-inflammatory agents (e.g.corticosteroids and non-steroidal anti-inflammatory agents (NSAIDs),other muscarinic receptor antagonists (i.e., anticholinergic agents),antiinfective agents (e.g. antibiotics or antivirals) andantihistamines. The other therapeutic agents can be used in the form ofpharmaceutically acceptable salts or solvates. Additionally, ifappropriate, the other therapeutic agents can be used as optically purestereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, bitolterol,pirbuterol, levalbuterol and the like, or pharmaceutically-acceptablesalts thereof. Other β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)hexyl]oxy}butyl)benzensulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422, published on Aug. 29,2002;3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490, published Sep. 12,2002;3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds disclosed in WO 02/076933, published on Oct. 3,2002;4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439, published on Mar. 27,2003;N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 B1, issued onJun. 10, 2003;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 B2, issued onNov. 25, 2003; and pharmaceutically-acceptable salts thereof. Whenemployed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative corticosteroids that can be used in combination with thecompounds of this invention include, but are not limited to, methylprednisolone, prednisolone, dexamethasone, fluticasone propionate, 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl)ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the corticosteroid will be present in the pharmaceuticalcomposition in a therapeutically effective amount. Typically, thesteroidal anti-inflammatory agent will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g. monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g. adenosine 2a agonists); cytokineantagonists (e.g. chemokine antagonists such as, an interleukin antibody(αIL antibody), specifically, an αIL-4 therapy, an αIL-13 therapy, or acombination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically-acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine(d,l)hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methantheline, propantheline bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate(Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride(Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically-acceptable saltthereof; or, for those compounds listed as a salt, alternatepharmaceutically-acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically-acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically-acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 μg/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 250 mg Lactose(spray-dried) 200 mg Magnesium stearate  10 mg

-   -   Representative Procedure: The ingredients are throughly blended        and then loaded into a hard gelatine capsule (460 mg of        composition per capsule).

Formulation Example B

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 20 mg Starch 89 mgMicrocrystalline cellulose 89 mg Magnesium stearate  2 mg

-   -   Representative Procedure: The ingredients are throughly blended        and then passed through a No. 45 mesh U.S. sieve and loaded into        a hard gelatin capsule (200 mg of composition per capsule).

Formulation Example C

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 100 mg Polyoxyethylenesorbitan monooleate  50 mg Starch powder 250 mg

-   -   Representative Procedure: The ingredients are throughly blended        and then loaded into a gelatin capsule (300 mg of composition        per capsule).

Formulation Example D

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention  10 mg Starch  45 mgMicrocrystalline cellulose  35 mg Polyvinylpyrrolidone (10 wt. % inwater)   4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5mg Talc   1 mg

-   -   Representative Procedure: The compound of the invention, starch        and cellulose are passed through a No. 45 mesh U.S. sieve and        mixed throughly. The solution of polyvinylpyrrolidone is mixed        with the resulting powders, and this mixture is then passed        through a No. 14 mesh U.S. sieve. The granules so produced are        dried at 50–60° C. and passed through a No. 18 mesh U.S. sieve.        The sodium carboxymethyl starch, magnesium stearate and talc        (previously passed through a No. 60 mesh U.S. sieve) are then        added to the granules. After mixing, the mixture is compressed        on a tablet machine to afford a tablet weighing 100 mg.

Formulation Example E

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 250 mg Microcrystallinecellulose 400 mg Silicon dioxide fumed  10 mg Stearic acid  5 mg

-   -   Representative Procedure: The ingredients are throughly blended        and then compressed to form tablets (665 mg of composition per        tablet).

Formulation Example F

Single-scored tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 400 mg Cornstarch  50 mgCroscarmellose sodium  25 mg Lactose 120 mg Magnesium stearate  5 mg

-   -   Representative Procedure: The ingredients are throughly blended        and compressed to form a single-scored tablet (600 mg of        compositions per tablet).

Formulation Example G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention  1.0 g Fumaric acid  0.5 gSodium chloride  2.0 g Methyl paraben  0.15 g Propyl paraben  0.05 gGranulated sugar  25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.)  1.0 g Flavoring 0.035 mL Colorings  0.5 mg Distilledwater q.s. to 100 mL

-   -   Representative Procedure: The ingredients are mixed to form a        suspension containing 100 mg of active ingredient per 10 mL of        suspension.

Formulation Example H

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 mg Lactose  25 mg

-   -   Representative Procedure: The compound of the invention is        micronized and then blended with lactose. This blended mixture        is then loaded into a gelatin inhalation cartridge. The contents        of the cartridge are administered using a powder inhaler.

Formulation Example I

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a bulk formulation ratio of micronized compound of the inventionto lactose of 1:200. The composition is packed into a dry powderinhalation device capable of delivering between about 10 and about 100μg of the compound of the invention per dose.

Formulation Example J

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt. % of a compoundof the invention and 0.1 wt. % lecithin is prepared by dispersing 10 gof the compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

Formulation Example K

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5% compound of theinvention, 0.5% lecithin, and 0.5% trehalose is prepared by dispersing 5g of active ingredient as micronized particles with mean size less than10 μm in a colloidal solution formed from 0.5 g of trehalose and 0.5 gof lecithin dissolved in 100 mL of demineralized water. The suspensionis spray dried and the resulting material is micronized to particleshaving a mean diameter less than 1.5 μm. The particles are loaded intocanisters with pressurized 1,1,1,2-tetrafluoroethane.

Formulation Example L

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.1 mg of the compound of theinvention in 1 mL of a 0.9% sodium chloride solution acidified withcitric acid. The mixture is stirred and sonicated until the activeingredient is dissolved. The pH of the solution is adjusted to a valueof about 5 by the slow addition of NaOH.

Formulation Example M

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4 M) 2.0 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and        the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.        Utility

The substituted pyrrolidine and related compounds of this invention areuseful as muscarinic receptor antagonists and therefore, such compoundsare useful for treating medical conditions mediated by muscarinicreceptors, i.e., medical conditions which are ameliorated by treatmentwith a muscarinic receptor antagonist. Such medical conditions include,by way of example, respiratory tract disorders, such as chronicobstructive pulmonary disease, asthma, pulmonary fibrosis, allergicrhinitis, rhinorrhea; genitourinary tract disorders, such as overactivebladder or detrusor hyperactivity and their symptoms; gastrointestinaltract disorders, such as irritable bowel syndrome, diverticular disease,achalasia, gastrointestinal hypermotility disorders and diarrhea;cardiac arrhythmias, such as sinus bradycardia; Parkinson's disease;cognitive disorders, such as Alzheimer's disease; dismenorrhea; and thelike.

In one embodiment, the compounds of this invention are useful fortreating smooth muscle disorders in mammals, including humans and theircompanion animals (e.g., dogs, cats etc.). Such smooth muscle disordersinclude, by way of illustration, overactive bladder, chronic obstructivepulmonary disease and irritable bowel syndrome.

When used to treat smooth muscle disorders or other conditions mediatedby muscarinic receptors, the compounds of this invention will typicallybe administered orally, rectally, parenterally or by inhalation in asingle daily dose or in multiple doses per day. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by the patient's physician and will dependon such factors as the nature and severity of the patients condition,the condition being treated, the age and general health of the patient,the tolerance of the patient to the active agent, the route ofadministration and the like.

Typically, suitable doses for treating smooth muscle disorders or otherdisorders mediated by muscarinic receptors will range from about 0.14μg/kg/day to about 7 mg/kg/day of active agent; including from about0.15 μg/kg/day to about 5 mg/kg/day. For an average 70 kg human, thiswould amount to about 10 μg per day to about 500 mg per day of activeagent.

In a specific embodiment, the compounds of this invention are useful fortreating respiratory disorders, such as COPD or asthma, in mammalsincluding humans. When used to treat respiratory disorders, thecompounds of this invention will typically be administered by inhalationin multiple doses per day, in a single daily dose or a single weeklydose. Generally, the dose for treating a respiratory disorder will rangefrom about 10 μg/day to about 200 μg/day.

When used to treat a respiratory disorder, the compounds of thisinvention are optionally administered in combination with othertherapeutic agents such as a β₂-adrenoreceptor agonist; acorticosteroid, a non-steroidal anti-inflammatory agent, or combinationsthereof.

In another embodiment, the compounds of this invention are used to treatoveractive bladder. When used to treat overactive bladder, the compoundsof this invention will typically be administered orally in a singledaily dose or in multiple doses per day; preferably in a single dailydose. Preferably, the dose for treating overactive bladder will rangefrom about 1.0 mg/day to about 500 mg/day.

In yet another embodiment, the compounds of this invention are used totreat irritable bowel syndrome. When used to treat irritable bowelsyndrome, the compounds of this invention will typically be administeredorally or rectally in a single daily dose or in multiple doses per day.Preferably, the dose for treating irritable bowel syndrome will rangefrom about 1.0 mg/day to about 500 mg/day.

Since compounds of this invention are muscarinic receptor antagonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having muscarinic receptors. Suchbiological systems or samples may comprise M₁, M₂, M₃, M₄ and/or M₅muscarinic receptors. Any suitable biological system or sample havingmuscarinic receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and thelike.

In this embodiment, a biological system or sample comprising amuscarinic receptor is contacted with a muscarinic receptor-antagonizingamount of a compound of this invention. The effects of antagonizing themuscarinic receptor are then determined using conventional proceduresand equipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTPγS for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). A compound of this invention will antagonize or decreasethe activation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. A muscarinicreceptor-antagonizing amount of a compound of this invention willtypically range from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for discovering new compounds that have muscarinic receptorantagonist activity. In this embodiment, muscarinic receptor bindingdata (for example, as determined by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe muscarinic receptor binding data for a compound of this invention toidentify those test compounds that have about equal or superiormuscarinic receptor binding, if any. This aspect of the inventionincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

Among other properties, compounds of this invention have been found tobe potent inhibitors of M₃ muscarinic receptor activity. Accordingly, inspecific embodiments, this invention is directed to compounds of formulaI having an inhibition dissociation constant for the M₃ receptor subtypeof less than or equal to 100 nM; or less than or equal to 50 nM; or lessthan or equal to 10 nM (as determined by in vitro radioliganddisplacement assays).

Additionally, compounds of this invention have also been found topossess a surprising and unexpected duration of bronchoprotection whenadministered by inhalation. Accordingly, in another specific embodiment,this invention is directed to compounds of formula I having a durationof bronchoprotection greater than about 24 hours, including about 24hours to about 72 hours, when administered by inhalation. The term“duration of bronchoprotection” means the length of time that a compoundprovides a bronchoprotective effect in the guinea pig model ofacetylcholine-induced bronchoconstriction.

Moreover, compounds of this invention have been found to possesssurprising and unexpected lung selectivity when administered byinhalation. Accordingly, in another specific embodiment, this inventionis directed to compounds of formula I having an apparentlung-selectivity index greater than 10 at either 1.5 hours or 24 hourspost-dosing by inhalation. The term “apparent lung-selectivity index”means either (a) the ratio of the anti-sialagogue ID₅₀ (dose required toinhibit pilocaripine-induced salivation by 50%) to the bronchoprotectiveID₅₀ (dose required to inhibit acetylcholine-induced bronchoconstrictionby 50%); or (b) the ratio of the anti-depressor ID₅₀ (dose required toinhibit methacholine-induced decrease in mean arterial pressure by 50%)to the bronchoprotective ID₅₀ (dose required to inhibitacetylcholine-induced bronchoconstriction by 50%).

These properties, as well as the utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeaning.

AC = adenylyl cyclase ACN = acetonitrile BSA = bovine serum albumin BOC= tert-butoxycarbonyl cAMP = cyclic adenosine monophosphate CHO =Chinese hamster ovary cpm = counts per minute DCM = dichloromethaneDIPEA = diisopropylethylamine DME = ethylene glycol dimethyl ether DMF =N,N-dimethylformamide DMSO = dimethyl sulfoxide dPBS = Dulbecco'sphosphate buffered saline, without CaCl₂ and MgCl EDC =1-(3-dimethylaminopropyl)-3- ethylcarbodimide hydrochloride EDDA =ethylenediaminetetraacetic acid EtOAc = ethyl acetate FBS = fetal bovineserum GDP = guanosine 5′-diphosphate HEPES =4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid hM₁ = humanmuscarinic receptor subtype 1 hM₂ = human muscarinic receptor subtype 2hM₃ = human muscarinic receptor subtype 3 hM₄ = human muscarinicreceptor subtype 4 hM₅ = human muscarinic receptor subtype 5 HOAT =1-hydroxy-7-azabenzotriazole HPLC = high performance liquidchromatography K_(i) = inhibition dissociation constant MS = massspectrometry MTBE = methyl tert-butyl ether [³H]NMS =l-[N-methyl-³H]scopolamine methyl chloride OIS = oxotremorine-inducedsalivation PMB = p-methoxybenzyl PyBOP =benzotriazol-1-yloxytripyrrolidino- phosphonium hexafluorophosphate THF= tetrahydrofuran TLC = thin layer chromatography TFA = trifluoroaceticacid

All temperatures reported in the following examples are in degreesCelsius (° C.) unless otherwise indicated. Also, unless noted otherwise,reagents, starting materials and solvents were purchased from commercialsuppliers (such as Aldrich, Fluka, Sigma and the like) and were usedwithout further purification.

Example A Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetaniideStep A—Preparation of (S)-1-Benzyl-3-(p-toluenesulfonyloxy)pyrrolidine

To a stirred solution of (S)-1-benzyl-3-pyrrolidinol (44.3 g, 0.25 mol)and 1,4-diazabicyclo[2.2.2]octane (33.7 g, 0.3 mol) in 250 mL oftert-butyl methyl ether under an atmosphere of nitrogen at 0° C., wasadded p-toluenesulfonyl chloride (52.4 g, 0.275 mol) portion-wise over20 min. The reaction mixture was stirred at 0° C. for 1 h. The ice bathwas removed and the mixture was stirred at ambient temperature overnight(20±5 h). Ethyl acetate (100 mL) was added, followed by saturatedaqueous sodium bicarbonate solution (250 mL). The resulting mixture wasstirred at ambient temperature for 1 h. The layers were separated andthe organic layer was washed with saturated aqueous sodium bicarbonatesolution (250 mL); saturated aqueous ammonium chloride solution (250mL); saturated aqueous sodium chloride solution (250 mL); and then driedover sodium sulfate (80 g). The sodium sulfate was filtered off andwashed with ethyl acetate (20 mL) and the solvent was removed in vacuoto give 78.2 g of the title intermediate as an off-white solid (94%yield; 95% purity by HPLC).

Step B—Preparation of(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)-pyrrolidine

To a stirred solution of diphenylacetonitrile (12.18 g, 61.8 mmol) inanhydrous THF (120 mL) at 0° C., potassium tert-butoxide (10.60 g, 94.6mmol) was added over 5 min. The reaction mixture was stirred at 0° C.for 1 h. To the reaction mixture at 0° C. was added(S)-1-benzyl-3-(p-toluenesulfonyloxy)-pyrrolidine (20.48 g, 61.3 mmol)in one portion. The cold bath was removed and the reaction mixture wasstirred for 5 to 10 min at which time the reaction mixture had become abrown homogeneous solution. The reaction mixture was then heated at 40°C. overnight (20±5 h). The reaction mixture (bright yellow suspension)was allowed to cool to room temperature before adding water (150 mL).Most of the THF was then removed in vacuo and isopropyl acetate (200 mL)was added. The layers were separated and the organic layer was washedwith saturated aqueous ammonium chloride solution (150 mL); saturatedaqueous sodium chloride solution (150 mL); and then dried over sodiumsulfate (50 g). The sodium sulfate was filtered off and washed withisopropyl acetate (20 mL) and the solvent was removed in vacuo to give23.88 g of the title intermediate as a light brown oil (>99% yield, 75%purity by HPLC, contaminated mainly with excess diphenylacetonitrile).

Step C—Preparation of (S)-3-(1-Cyano-1,1-diphenylmethyl)pyrrolidine

(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine was dissolved inisopropyl acetate (ca. 1 g/10 mL) and the solution was mixed with anequal volume of 1N aqueous hydrochloric acid. The resulting layers wereseparated and the aqueous layer was extracted with an equal volume ofisopropyl acetate. The organic layers were combined, dried over sodiumsulfate and filtered. The solvent was removed in vacuo to afford(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride asa light yellow foamy solid. (Note: This hydrochloride salt can also beprepared during the work-up of Step B).

To a stirred solution of(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride(8.55 g, 21.98 mmol) in methanol (44 mL) was added palladium on carbon(1.71 g) and ammonium formate (6.93 g, 109.9 mmol). The reaction mixturewas heated to 50° C. with stirring for 3 h. The reaction was cooled toambient temperature and water (20 mL) was added. The resulting mixturewas filtered through a pad of Celite, washing with methanol (20 mL). Thefiltrate was collected and most of the methanol was removed in vacuo.The residue was mixed with isopropyl acetate (100 mL) and 10% aqueoussodium carbonate (50 mL). The resulting layers were separated and theaqueous layer was extracted with isopropyl acetate (50 mL). The organiclayers were combined and dried over sodium sulfate (20 g). The sodiumsulfate was filtered off and washed with isopropyl acetate (20 mL). Thesolvent was removed in vacuo to afford 5.75 g of the title intermediateas a light yellow oil (99.7% yield, 71% purity by HPLC).

Step D—Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide

A 200 mL flask with a magnetic stir bar and a nitrogen inlet was chargedwith (S)-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine (2.51 g) and 80%H₂SO₄ (19.2 mL; pre-prepared with 16 mL of 96% H₂SO₄ and 3.2 mL of H₂O).The reaction mixture was then heated at 90° C. for 24 h or untilstarting material was consumed as indicated by HPLC. The reactionmixture was allowed to cool to room temperature and then poured onto ice(ca. 50 mL by volume). A 50% aqueous sodium hydroxide solution was addedslowly to the mixture with stirring over an ice bath until the pH wasabout 12. Dichloromethane (200 mL) was added and mixed with the aqueoussolution at which time sodium sulfate precipitated out and was filteredoff. The filtrate was collected and the layers were separated. Theaqueous layer was extracted with dichloromethane (100 mL) and theorganic layers were combined and dried with over sodium sulfate (5 g).The sodium sulfate was filtered off and washed with dichloromethane (10mL). The solvent was removed in vacuo to give the crude product as alight yellow foamy solid (ca. 2.2 g, 86% purity by HPLC).

The crude product was dissolved in ethanol (18 mL) with stirring. Tothis solution was added a warm solution of L-tartaric acid (1.8 g) inethanol (14 mL) and the resulting mixture was stirred overnight (15±5h). The resulting precipitate was isolated by filtration to give anoff-white solid (ca. 3.2 g, >95% purity by HPLC). Methanol (15 mL) wasadded to this solid and the resulting slurry was stirred at 70° C.overnight (15 h). The slurry was allowed to cool to ambient temperatureand a white solid (˜2.6 g, >99% purity by HPLC) was obtained afterfiltration. To this solid was added ethyl acetate (30 mL) and 1 Naqueous sodium hydroxide (25 mL). This mixture was mixed until twodistinct layers formed and then the layers were separated and theaqueous layer was extracted with ethyl acetate (20 mL). The organiclayers were combined and dried over sodium sulfate (10 g). The sodiumsulfate was removed by filtration and the solvent was evaporated invacuo to afford 1.55 g of the title intermediate as an off-white foamysolid (58% yield; >99% purity by HPLC).

Example 1 Synthesis of2-[(S)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide StepA—Preparation of 8-(N-Benzyl-N-methylamino)octan-1-ol

8-Bromo-1-octanol (25 g, 119.6 mmol) in acetonitrile (50 mL) was addedto a stirred solution of N-benzyl-N-methylamine (43.49 g, 358.9 mmol)and potassium carbonate (49.52 g, 358.9 mmol) in acetonitrile (250 mL)at 35° C. The reaction mixture was then stirred at 35° C. for 7 h andthen cooled to ambient temperature. The potassium carbonate was filteredand the filtrate was concentrated under reduced pressure. The cruderesidue was dissolved in MTBE (400 mL) and the organic phase was washedwith water, brine and dried over magnesium sulfate.N-methyl-2-pyrrolidone was added and the mixture was concentrated underreduced pressure to remove excess N-benzyl-N-methylamine. MTBE (400 mL)was added and the organic phase was washed with water, brine, and driedover magnesium sulfate, filtered and concentrated under reduced pressureto afford the title intermediate as an oil (˜100% conversion).

Analytical Data: MS m/z 250.3 (MH⁺).

Step B—Preparation of 8-(N-Benzyl-N-methylamino)octanal

Dimethylsulfoxide (22.71 mL, 320 mmol) and then diisopropylethylamine(55.74 mL, 320 mmol) were added to a stirred solution of theintermediate from Step A (20 g, 80 mmol) in dichloromethane (200 mL) at−10° C. The reaction mixture was stirred at −10° C. for 30 min, thensulfur trioxide pyridine complex (38 g, 240 mmol) was added portionwise.The reaction mixture was stirred for an additional 1 h at −10° C. andthen water (200 mL) was added. The organic layer was separated andwashed with water (200 mL), brine (30 mL), dried over magnesium sulfateand then concentrated under reduced pressure. Toluene (100 mL) was addedand removed under reduced pressure to afford the title intermediate asoil (˜100% conversion).

Step C—Preparation of2-[(S)-1-(8-N-Benzyl-N-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A solution of the intermediate from Step B (5.95 g, 24 mmol) and2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (7.4 g, 26.4 mmol) indichloromethane (250 mL) was cooled at 0° C. and stirred for 10 min.Sodium triacetoxyborohydride (8.4 g, 36 mmol) was added portionwise at0° C. and the reaction mixture was stirred at room temperature for 4 h.Dichloromethane was added and the organic phase was washed with sodiumbicarbonate (2×), brine (1×), dried over magnesium sulfate andconcentrated under reduced pressure. The crude product was purified byflash chromatography (DCM/MeOH/NH₄OH=90/9/1) to give 9 g of the titleintermediate as an oil (75% yield).

Analytical Data: MS m/z 512.8 (MH⁺).

Step D—Preparation of2-[(S)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

To a stirred solution of the intermediate from Step C (9 g, 17.6 mmol)in acetic acid (170 mL) under a nitrogen atmosphere was added palladiumon carbon (10 wt. %, 600 mg) and palladium hydroxide on carbon (20 wt.%, wet, 600 mg). The reaction mixture was flushed with nitrogen threetimes and then placed under a hydrogen-containing balloon for 3 days atroom temperature. The reaction mixture was filtered through Celite,washing with acetic acid, and the solvent was removed under reducedpressure. The resulting residue was purified by prep HPLC to afford 4.02g of the title compound as its bis-trifluoroacetic acid salt, which asan oil (35% yield).

Analytical Data: MS m/z 422.2 (MH⁺).

Alternatively, the title compound was prepared as follows:

Step A—Preparation of 8-(N-Benzyl-N-methylamino)octan-1-ol

From 8-Bromooctan-1-ol: To a 250 mL flask was chargedN-benzyl-N-methylamine (24.3 g, 200 mmol), potassium carbonate (28 g,200 mmol), 8-bromooctan-1-ol (14 g, 67 mmol) and acetonitrile (150 mL).This reaction mixture was stirred at 35–40° C. for 5 h. The solidmaterial was then filtered and the filtrate was distilled to an oilunder high vacuum to remove excess N-benzyl-N-methylamine. The residuewas dissolved in 150 mL of MTBE and washed with 15% ammonium chloridesolution (2×100 mL), brine (100 mL), dried with 20 g of sodium sulfate,filtered and distilled under vacuum to give 13.2 g of the titleintermediate as an oil (79% yield).

From 8-Chlorooctan-1-ol: A 2-L flask was charged with benzylmethylamine(270 g, 2.23 mol), sodium carbonate (157 g, 1.48 mol), sodium iodide(11.1 g, 0.074 mol), 8-chlorooctanol (122 g, 0.74 mol) and acetonitrile(1000 mL) and the resulting suspension was stirred at 80° C. for 20–30h. The reaction mixture was then concentrated to a volume of about 500mL and water (600 mL) and tert-butyl methyl ether (1000 mL) were added.The MTBE layer was then separated and washed with water (500 mL). TheMTBE solution was concentrated by distillation under vacuum to providean oil and the oil was then further concentrated by distillation underhigh vacuum to remove excess benzylmethylamine. N-methyl-2-pyrrolidone(300 mL) was then added to the remaining oil and this solutionconcentrated by distillation under high vacuum to provide an oil. Theoil was dissolved in MTBE (1000 mL) and the resulting solution waswashed with water (2×500 mL), brine (500 mL), dried with sodium sulfate(100 g), filtered and concentrated by distillation to afford the titlecompound as an oil (178 g, 96% yield, >95% purity).

Step B—Preparation of Toluenesulfonic Acid8-(N-Benzyl-N-methylamino)octan-1-yl Ester

A 250 mL flask was charged with the intermediate from Step A (10 g),DABCO (6.72 g), and MTBE (100 ml). The reaction mixture was cooled to<10° C. and a solution of toluenesulfonic chloride (9.2 g) in 60 mL ofMTBE was added at <15° C. This reaction mixture was stirred at roomtemperature for 2 h and then heptane (40 mL) was added and the mixturewas filtered. The filtrate was distilled under vacuum to give 16 g ofthe title intermediate as an oil (99% yield).

Step C—Preparation of2-[(S)-1-(8-N-Benzyl-N-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A 1000 mL flask with a nitrogen inlet was charged with the intermediatefrom Step B (16 g), 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (10 g),diisopropylethylamine (10.3 g) and acetonitrile (200 mL). The reactionmixture was stirred at 45–50° C. for 20 h and then acetic anhydride (2g) was added and the mixture stirred at room temperature for 2 hours.tert-Butyl methyl ether (300 mL) and water (400 mL) were added and theMTBE layer was separated and washed with water (2×150 mL) and then 1NHCl (1×150 mL). The aqueous layer was separated and washed with MTBE(3×100 mL) and then made basic with 27% ammonium hydroxide solution topH>12. The basic aqueous layer was then extracted with MTBE (2×200 mL)and the MTBE layer was washed with water (200 mL), brine (200 mL), driedover sodium sulfate (20 g), filtered and distilled to give 16.5 g of thetitle intermediate as an oil (90% yield). If desired, this reaction canbe conducted in N-methylpyrrolidone as the solvent. Additionally,potassium carbonate or sodium carbonate can be used in place ofdiisopropylethylamine and optionally sodium iodide may be added to thereaction mixture.

Step D—Preparation of2-[(S)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A 250 mL flask was charged with the intermediate from Step C (24 g),palladium on carbon (10% palladium on carbon with 50% water, 5.3 g)),isopropanol (160 mL) and 3 M HCl solution (30 mL). The reaction mixturewas degassed with nitrogen and then was hydrogenated (45–50 psi) at roomtemperature for 16 h. The mixture was then filtered though a Celite padand the filtrate was distilled to a volume of about 50 mL. The residuewas dissolved in 1 N HCl (100 mL) and washed with dichloromethane (2×100mL). The aqueous layer was adjusted to pH>12 by adding ammoniumhydroxide and then extracted using MTBE (2×150 mL). The MTBE solutionwas then washed with water (100 mL), brine (100 mL), dried over sodiumsulfate (30 g), filtered and distilled to oil which was dried under highvacuum to give 16.5 g of the title compound (91% yield).

Alternatively, the title compound was prepared as follows:

Step A—Preparation of 8,8-Dimethoxyoctanal

A 1 L flask was charged cyclooctene (50 g), methanol (250 mL) anddichloromethane (250 mL). Ozone was bubbled into the solution at−70° C.for 8 h. Toluenesulfonic acid (3 g) was then added and the reactionmixture was stirred at −70° C. for 6 h. Sodium bicarbonate (20 g) wasthen added and the reaction mixture stirred for an additional 2 h at−60° C. Finally, dimethyl sulfite (56 g) was added at −60° C. and thereaction mixture was stirred at room temperature for 16 h. The solidthat had formed was filtered and filtrate was evaporated to oil. The oilwas dissolved in dichloromethane (300 mL) and washed with 1% sodiumbicarbonate solution (2×150 mL). The dichloromethane solution was thendried over sodium sulfate (50 g), filtered and distilled to give 60.3 gof the title intermediate as an oil (71% yield).

Step B—Preparation of2-[(S)-1-(8-Oxooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A 100 mL flask was charged with2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (2.8 g),8,8-dimethoxyoctanal (2.1 g), and dichloromethane (20 mL) and thismixture was stirred at room temperature for 1 h. Sodiumtriacetoxyborohydride (3.18 g) was added and the reaction mixture wasstirred at room temperature for 14 h. A solution of 5% sodiumbicarbonate (350 mL) was then added and this mixture stirred for 0.5hours. The layers were separated and the aqueous layer was extractedwith dichloromethane (20 mL). The combined dichloromethane solution wasconcentrated to a volume of about 20 mL, filtered through a silica gelpad (10 g) and washed with 10% methanol in dichloromethane (100 mL). Theproduct solution was concentrated to an oil and the oil was dissolved in50 mL of acetonitrile and stirred with 1% HCl (30 mL) for 16 hours. Themixture was made basic to approximately pH>12 by adding 28% ammoniumhydroxide solution and then extracted with MTBE (2×100 mL). The MTBElayer was washed with brine (100 mL), dried over sodium sulfate (10 g),filtered and concentrated under vacuum to give 3.8 g of the titleintermediate as an oil (93% yield).

Step C—Preparation of2-[(S)-1-(8-N-Benzyl-N-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A 100 mL flask with a nitrogen inlet was charged with the intermediatefrom Step B (3 g), N-benzyl-N-methylamine (2.1 g) and dichloromethane(20 mL) and this mixture was stirred at room temperature for 1 h. Sodiumtriacetoxyborohydride (3.18 g) was added and the reaction mixture wasstirred at room temperature for 14 h. The reaction was then quenched byadding 50 mL of 5% HCl and the resulting mixture was stirred for 0.5hours. The layers were separated and the aqueous layer was washed withdichloromethane (20 mL). The aqueous layer was adjusted to pH>13 byadding 50% potassium hydroxide and extracted with MTBE (2×100 mL). Thecombined MTBE solution was washed with brine (100 ml), dried with sodiumsulfate (10 g), filtered and concentrated to give 2.8 g of the titleintermediate as an oil (75% yield). Using the procedure described inStep D above, this intermediate was converted into the title compound.

Alternatively, the title compound was prepared as thenaphthalene-1,5-disulfonic acid salt using the following procedure:

Step A—Preparation of 8-(N-tert-Butoxycarbonyl-N-methylamino)octan-1-ol

A 1-L flask was charged with 8-(benzylmethylamino)octan-1-ol (49 g, 0.20mol), isopropanol (400 mL), 2 N aqueous hydrochloric acid (100 mL) andactivated carbon (5 g, DARCO) and the resulting mixture was stirred for30 minutes. The mixture was then filtered to remove the activated carbonand to the filtrate was added palladium on carbon (5 g, 10% dry weight).The resulting mixture was degassed three times with nitrogen and thentwice with hydrogen; and then the mixture was hydrogenated on a Parrshaker at 20–30 psi hydrogen for 12–24 hours. The mixture was thenfiltered through a 20 g pad of Celite and concentrated by distillationto a volume of about 100 mL. Isopropanol (200 mL) was added and thissolution was again concentrated by distillation under vacuum to a volumeof about 100 mL. This procedure was repeated two more times to give asolution containing 8-methylaminooctan-1-ol hydrochloride.

A 1-L flask was charged with the 8-methylaminooctanol hydrochlorideisopropanol solution from above and triethylamine (30.3 g, 0.30 mol),and to this mixture was added di-tert-butyl dicarbonate (48 g, 0.22 mol)in portions. The resulting mixture was stirred at room temperature for2–5 h and then the mixture was concentrated to a volume of about 300 mL.Water (200 mL) and ethyl acetate (400 mL) were added and this mixturewas stirred for 15 minutes. The organic layer was then separated andwashed with water (300 mL), brine (300 mL), dried over Na₂SO₄ (50 g),filtered and solvent reduced under vacuum to afford the title compoundas a light yellow oil. (40 g, 77% yield, ˜95% purity).

Step B—Preparation of Toluene-4-sulfonic Acid8-(N-tert-Butoxycarbonyl-N-methylamino)octyl Ester

In a 250 mL flask, a solution of the product from Step A (5.2 g, 20mmol) and DABCO (3.13 g, 2.8 mmol) in MTBE (30 mL) was cooled to about10° C. and a solution of p-toluenesulfonyl chloride (4.2 g, 22 mmol) inMTBE (20 mL) was added while maintaining the temperature of the reactionmixture at 20° C. or less. The resulting solution was then stirred atroom temperature for 2 h. Water (100 mL) was added and the mixture wasstirred for 15 minutes. The organic layer was separated, washed withwater (100 mL), brine (100 mL) and then concentrated by distillation togive the title compound as an oil.

Step C—Preparation of2-{(S)-1-[8-(N-tert-Butoxycarbonyl-N-methylamino)octyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

To a 500 mL flask was added the product from Step B (17.68 g, 43 mmol),the product from Preparation 1 (12 g, 43 mmol), diisopropylethylamine(16.55 g, 128 mmol) and acetonitrile (100 mL). The resulting mixture wasstirred at 60° C. to 65° C. for 5 to 7 hours and then cooled to roomtemperature. The solvent was reduced in vacuo and isopropyl acetate (100mL) was added to dissolve the residue. The resulting solution was washedwith water (100 mL), saturated NaHCO₃ solution (100 mL), brine (100 mL),dried over MgSO₄ (5 g) and filtered to afford an orange solution.

A silica gel (115 g, 280–400 mesh) pad was pre-treated with 400 mL ofisopropyl acetate containing 1% triethylamine, following by 250 mL ofisopropyl acetate (the silica gel pad is about 6.4 cm in diameter andabout 10.2 cm in height). The filtrate from above (about 150 mL involume) was loaded directly onto the pre-treated silica pad and elutedwith isopropyl acetate (400 mL) and then with 20% isopropanol inisopropyl acetate (1000 mL). The product fractions were combined andconcentrated to afford the title compound as an oil (17.16 g, 77% yield,97% purity).

Step D—Preparation of2-[(S)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamideNalphthalene-1,5-disulfonic Acid Salt

To a 1000 mL flask was added the product from Step C (9.88 g, 19 mmol),1,5-naphthalenedisulfonic acid tetrahydrate (13.69 g, 38 mmol) andisopropanol containing 3% water (497 mL). This mixture was heated to 85°C. for 3 to 5 hours, then slowly cooled to room temperature over a 4hour period and then stirred at room temperature for 12 to 24 hours. Theresulting solid was filtered and washed with isopropanol containing 3%water by volume (400 mL) and dried under vacuum for 10 to 15 hours atroom temperature to give the title compound as a crystalline solid(12.59 g, 95% yield, ˜99% purity).

If desired, this salt can be further purified by the followingprocedure:

To an 1 L flask was added2-[(S)-1-(8-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamidenaphthalene-1,5-disulfonic acid salt (21.4 g, 30.1 mmol) and isopropanolcontaining 3% water by volume (637 mL). The resulting slurry was stirredat 80° C. for 2 hours and then slowly cooled to room temperature andthen stirred at room temperature for 12 hours. The resulting crystallinesalt was filtrated, washed with isopropanol (600 mL) and then driedunder vacuum and nitrogen for 16 hours at room temperature to give thetitle compound as a white, crystalline solid (20.4 g, 96% yield).

Example 2 Synthesis of2-[(S)-1-(8-Isopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamideStep A—Preparation of2-[(S)-1-(8-Hydroxyoctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

8-Bromo-1-octanol (2.51 g, 12 mmol) in acetonitrile (10 mL) was added toa stirred solution of 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (2.8g, 10 mmol) and triethylamine (4.27 mL, 30 mmol) in acetonitrile (90 mL)at 40° C. The reaction mixture was heated at 55° C. for 16 h and thencooled to ambient temperature. The solvent was then removed underreduced pressure. The crude residue was dissolved in ethyl acetate (100mL) and the organic phase was washed with saturated aqueous sodiumbicarbonate (50 mL), dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified byflash chromatography (eluent: DCM/MeOH/NH₄OH=90/9/1) to give 1.8 g ofthe title intermediate as oil (44% yield).

Step B—Preparation of2-[(S)-1-(8-Oxooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

Dimethylsulfoxide (1.57 mL, 22.1 mmol), followed bydiisopropylethylamine (3.85 mL, 22.1 mmol) was added to a stirredsolution of the intermediate from Step A (1.8 g, 4.4 mmol) indichloromethane (44 mL) at 0° C. The reaction mixture was stirred at−10° C. for 15 min and then sulfur trioxide pyridine complex (2.1 g,13.2 mmol) was added. The reaction mixture was stirred for a further 2 hat −10° C. Water (50 mL) and DCM(50 ml) were added and the organic layerwas separated and washed with saturated aqueous sodium bicarbonate (2×30mL), saturated aqueous copper (II) sulfate solution (2×15 mL) and brine(30 mL). The organic layer was then dried over magnesium sulfate andconcentrated under reduced pressure to afford 1.5 g of the titleintermediate as oil (84% yield).

Step C—Preparation of2-[(S)-1-(8-Isopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

The intermediate from Step B (40.6 mg, 0.1 mmol) and isopropylamine(10.2 μL, 0.12 mmol) in 1,2-dichloroethane (1 mL) were stirred at roomtemperature for 1 h and then sodium triacetoxyborohydride (35.1 mg, 1.5mmol) was added. The reaction mixture was stirred for 16 h and then thesolvent was removed under reduced pressure. The residue was purified byHPLC to afford the title compound as its bis-trifluoroacetic acid salt.

Analytical Data: MS m/z 450.3 (MH⁺).

Using the procedures described herein and the appropriate startingmaterials, the compounds shown in Table IV were prepared:

TABLE IV Ex. Compound MS¹ 32-[(S)-1-(8-Prop-1-ylaminooctyl)pyrrolidin-3-yl]-2,2- 450.2diphenylacetamide 42-[(S)-1-(8-Cyclopropylaminooctyl)pyrrolidin-3-yl]-2,2- 448.3diphenylacetamide 52-[(S)-1-(8-Cyclobutylaminooctyl)pyrrolidin-3-yl]-2,2- 462.2diphenylacetamide 6 2-[(S)-1-(8-Cyclopentylaminooctyl)pyrrolidin-3-yl]-2,2- 476.3diphenylacetamide 7 2-[(S)-1-(8-Ethylaminooctyl)pyrrolidin-3-yl]-2,2-436.2 diphenylacetamide 82-{(S)-1-[8-(2-Hydroxyethyl)aminooctyl]pyrrolidin-3-yl}-2,2- 452.2diphenylacetamide 92-{(S)-1-[8-(R)-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin- 466.33-yl}-2,2-diphenylacetamide 102-{(S)-1-[8-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin-3- 466.3yl}-2,2-diphenylacetamide 112-{(S)-1-[8-(S)-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin- 466.23-yl}-2,2-diphenylacetamide 122-{(S)-1-[8-(2,2,2-Trifluoroethyl)aminooctyl]pyrrolidin-3- 490.2yl}-2,2-diphenylacetamide 132-[(S)-1-(8-Benzylaminooctyl)pyrrolidin-3-yl]-2,2- 498.2diphenylacetamide 14 2-[(S)-1-(9-Methylaminnonyl)pyrrolidin-3-yl]-2,2-NA^(‡) diphenylacetamide 272-[(R)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2- 422.4diphenylacetamide ¹Mass Spectrometry: m/z (MH⁺). ^(‡)Not available.

Additionally, using the procedures described herein and the appropriatestarting materials, the compounds in Table V can be prepared:

TABLE V Ex. Compound 152-[(S)-1-(9-Isopropylaminononyl)pyrrolidin-3-yl]-2,2- diphenylacetamide16 2-[(S)-1-(9-Prop-1-ylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 172-[(S)-1-(9-Cyclopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 182-[(S)-1-(9-Cyclobutylaminononyl)pyrrolidin-3-yl]-2,2- diphenylacetamide19 2-[(S)-1-(9-Cyclopentylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 202-[(S)-1-(9-Ethylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 212-{(S)-1-[9-(2-Hydroxyethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 222-{(S)-1-[9-(R)-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 232-{(S)-1-[9-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 242-{(S)-1-[9-(S)-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 252-{(S)-1-[9-(2,2,2-Trifluoroethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 26 2-[(S)-1-(9-Benzylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 282-[(R)-1-(8-Isopropylaminooctyl)pyrrolidin-3-yl]-2,2- diphenylacetamide29 2-[(R)-1-(8-Prop-1-ylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 302-[(R)-1-(8-Cyclopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 312-[(R)-1-(8-Cyclobutylaminooctyl)pyrrolidin-3-yl]-2,2- diphenylacetamide32 2-[(R)-1-(8-Cyclopentylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 332-[(R)-1-(8-Ethylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 342-{(R)-1-[8-(2-Hydroxyethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 352-{(R)-1-[8-(R)-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 362-{(R)-1-[8-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 372-{(R)-1-[8-(S)-(1-Hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 382-{(R)-1-[8-(2,2,2-Trifluoroethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 392-[(R)-1-(8-Benzylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 402-[(R)-1-(9-Methylaminononyl)pyrrolidin-3-yl]-2,2- diphenylacetamide 412-[(R)-1-(9-Isopropylaminononyl)pyrrolidin-3-yl]-2,2- diphenylacetamide42 2-[(R)-1-(9-Prop-1-ylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 432-[(R)-1-(9-Cyclopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 442-[(R)-1-(9-Cyclobutylaminononyl)pyrrolidin-3-yl]-2,2- diphenylacetamide45 2-[(R)-1-(9-Cyclopentylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 462-[(R)-1-(9-Ethylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 472-{(R)-1-[9-(2-Hydroxyethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 482-{(R)-1-[9-(R)-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 492-{(R)-1-[9-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 502-{(R)-1-[9-(S)-(1-Hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 512-{(R)-1-[9-(2,2,2-Trifluoroethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 52 2-[(R)-1-(9-Benzylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 532-[1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 542-[1-(8-Isopropylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 552-[1-(8-Prop-1-ylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 562-[1-(8-Cyclopropylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 572-[1-(8-Cyclobutylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 582-[1-(8-Cyclopentylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 592-[1-(8-Ethylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 602-{1-[8-(2-Hydroxyethyl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide 612-{1-[8-(R)-(1-Hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide 622-{1-[8-(1-Hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide 632-{1-[8-(S)-(1-Hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide 642-{1-[8-(2,2,2-Trifluoroethyl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide 652-[1-(8-Benzylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide 662-[1-(9-Methylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide 672-[1-(9-Isopropylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide 682-[1-(9-Prop-1-ylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide 692-[1-(9-Cyclopropylaminononyl)piperidin-4-yl]-2,2- diphenylacetamide 702-[1-(9-Cyclobutylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide 712-[1-(9-Cyclopentylaminononyl)piperidin-4-yl]-2,2- diphenylacetamide 722-[1-(9-Ethylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide 732-{1-[9-(2-Hydroxyethyl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide 742-{1-[9-(R)-(1-Hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide 752-{1-[9-(1-Hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide 762-{1-[9-(S)-(1-Hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide 772-{1-[9-(2,2,2-Trifluoroethyl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide 782-[1-(9-Benzylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide

Comparative Example A Synthesis of2-[(S)-1-(8-Dimethylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

A 50 mL dry round bottom flask was charged with2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (200 mg, 0.714 mmol) andchloroform (20 mL), and then purged with nitrogen. Dimethylamine (535μL, 1.071 mmol) was added followed by the dropwise addition of1,8-dibromooctane (131 μL, 0.714 mmol). The reaction mixture was heatedto 50° C. and stirred for approximately 60 hours. The yellow homogeneousmixture was cooled to room temperature and extracted with 1.0 M aqueoushydrogen chloride that was then washed with fresh chloroform. To theacidic aqueous layer was added ethyl acetate and the mixture was madebasic to pH 13 with 10.0 M aqueous sodium hydroxide. The basic aqueouslayer was then extracted with additional ethyl acetate (2×15 mL). Thecombined organic layers were then washed with saturated aqueous sodiumchloride, dried over sodium sulfate, filtered and evaporated to give thecrude product. The crude product (223.0 mg) was purified by preparatoryHPLC and lyophilized to give the title compound as itsbis(trifluoroacetate) salt, which was a white hygroscopic solid.

Analytical Data: MS m/z 436.4 (C₂₈H₄₁N₃O+H)⁺; calc'd 436.3.

Comparative Example B Synthesis of2-[(S)-1-(9-Dimethylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

Using the procedures described herein and the appropriate startingmaterials, the title compound was prepared as its bis(trifluoroacetate)salt, which was a white hygroscopic solid.

Analytical Data: MS m/z 450.4 (C₂₉H₄₃N₃O+H)⁺; calc'd 450.3.

Comparative Example C Synthesis of2-{(S)-1-[8-N-(2-Hydroxyethyl)-N-methylaminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Using the procedures described herein and the appropriate startingmaterials, the title compound was prepared as its bis(trifluoroacetate)salt, which was a white hygroscopic solid.

Analytical Data: MS m/z 480.2; calc'd 480.4.

Comparative Example D Synthesis of2-[(S)-1-(8-Aminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide StepA—Preparation of2-[(S)-1-(8-Bromoooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

To a solution of 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (1.2 g,0.004 mol) and diisopropylethylamine (0.74 mL, 0.004 mol) in a 1:1 (v/v)mixture of acetone and DMF (20 mL) was added 1,8 dibromooctane (0.99 mL,0.005 mol). The mixture was heated to 40° C. for five hours and thenconcentrated to dryness and diluted with dichloromethane (20 mL). Theresulting mixture was washed with saturated sodium bicarbonate (2×20mL), brine (1×20 mL), dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography,eluting with 5% methanol/dichloromethane, to provide 315 mg of the titleintermediate as a white solid (15% yield).

Analytical Data: MS m/z 472.5 (MH⁺); calc'd 472.2.

Step B—Preparation of2-[(S)-1-(8-Di-tert-BOC-aminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

To a solution of di-tert-butyliminodicarboxylate (61 mg, 0.28 mmol) in 5mL of DMF at −10° C. as added sodium hydride (11 mg, 0.28 mmol; 60% inmineral oil). The solution was allowed to slowly warm to roomtemperature and after stirring for 2 hours the intermediate from Step A(0.095 mg, 0.20 mol) in dimethyl formamide (5 mL) was added. Thereaction mixture was then allowed to stir at room temperature overnightand then it was concentrated under vacuum, diluted with 10 mL ofdichloromethane and this mixture was washed with saturated sodiumbicarbonate (2×10 mL), brine (1×110 mL), dried over magnesium sulfate,filtered and concentrated to provide 120 mg of the title intermediate asa white solid. (99% yield).

Analytical Data: MS m/z 608.8 (MH⁺); calc'd 608.5.

Step C—Preparation of2-[(S)-1-(8-Aminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

To the intermediate from Step B (120 mg, 0.16 mmol) was added a mixtureof trifluoroacetic acid (0.08 mL) in dichloromethane (0.720 mL) and thereaction mixture was stirred at room temperature for four hours. Thereaction mixture was then concentrated to dryness under vacuum, dilutedwith dichloromethane (10 mL) and 1N sodium hydroxide was added slowlyuntil pH reached 14. The organic layer as separated and washed withsaturated sodium bicarbonate (2×10 mL), brine (1×110 mL), dried overmagnesium sulfate, filtered, concentrated. The residue was purified bypreparative HPLC to afford 27 mg of the title compound as itsbis-trifluoroacetic acid salt, which was a white solid.

Analytical Data: MS m/z 408.6 (MH⁺); calc'd 408.3.

Assay 1 Radioligand Binding Assay

A. Membrane Preparation from Cells Expressing hM₁, hM₂, hM₃ and hM₄Muscarinic Receptor Subtypes

CHO (Chinese hamster ovary) cell lines stably expressing cloned humanhM₁, hM₂, hM₃ and hM₄ muscarinic receptor subtypes, respectively, weregrown to near confluency in medium consisting of HAM's F-12 supplementedwith 10% FBS (Fetal Bovine Serum) and 250 μg/mL Geneticin. The cellswere grown in a 5% CO₂, 37° C. incubator and lifted with 2 mm EDTA indPBS. Cells were collected by 5 minute centrifugation at 650×g, and cellpellets were either stored frozen at −80° C. or membranes were preparedimmediately. For membrane preparation, cell pellets were resuspended inlysis buffer and homogenized with a Polytron PT-2100 tissue disrupter(Kinematica AG; 20 seconds×2 bursts). Crude membranes were centrifugedat 40,000×g for 15 minutes at 4° C. The membrane pellet was thenresuspended with resuspension buffer and homogenized again with thePolytron tissue disrupter. The protein concentration of the membranesuspension was determined by the method described in Lowry, O. et al.,1951, Journal of Biochemistry: 193, 265. All membranes were storedfrozen in aliquots at −80° C. or used immediately. Aliquots of preparedhM₅ receptor membranes were purchased directly from Perkin Elmer andstored at −80° C. until use.

B. Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂,hM₃, hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM1, hM2, hM3, hM4 or hM5 muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10–15 μg for hM₂, 10–20 μg for hM₃, 10–20 μgfor hM₄, and 10–12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 μM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mmHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed lC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W H. (1973) Biochemical Pharmacology,22(23):3099–108). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that a test compound has ahigher binding affinity for the receptor tested. The compound of formulaI was found to have a K_(i) value of about 0.96 nM for the M₃ muscarinicreceptor subtype in this assay.

Test compounds having a lower K_(i) value in this assay have a higherbinding affinity for the muscarinic receptor. The compounds of thisinvention which were tested in this assay had a K_(i) value for hM₂ranging from about 200 nM to less than 1 nM; typically ranging fromabout 100 nM to less than 1 nM; and a K_(i) value for hM₃ ranging fromabout 100 nM to less than 1 nM; typically ranging from about 50 nM toless than 1 nM. For example, the compounds of Examples 1–11, 14, 26, 27,and 39 had a K_(i) value for hM₃ of less than 50 nM. Thus, compounds ofthis invention were found to bind potently to the hM₂ and hM₃ receptorsubtypes in this assay.

Additionally, the binding affinity for compounds of the formula:

are shown in Table V (where R⁵, R^(x) and e are as defined in Table V):

TABLE V Compound hM₂ hM₃ Ex. No. R⁵ R^(x) e (nM) Δ^(†) (nM) Δ^(†) Ex. 1—CH₃ —H 8 3.8 0.96 Comp. Ex. A —CH₃ —CH₃ 8 18 ×4.7 2.5 ×2.6 Comp. Ex. D—H —H 8 150 ×39 50 ×52 Ex. 14 —CH₃ —H 9 1.7 0.66 Comp. Ex. B —CH₃ —CH₃ 9120 ×70 25 ×37 Ex. 8 —CH₂CH₂OH —H 8 13 5.1 Comp. Ex. C —CH₂CH₂OH —CH₃ 883 ×6.3 73 ×14 ^(†)Change in binding affinity relative to compound ofthe invention.

The data in Table V demonstrate that substitution of the terminal aminogroup with an additional alkyl group, such as methyl, significantlydecreases binding affinity at the hM₂ and hM₃ receptor subtypes.Additionally, the data in Table V demonstrate that removal of an alkylgroup, such as methyl, from the terminal amino group significantlydecreases binding affinity at the hM₂ and hM₃ receptor subtypes.

Assay 2 Muscarinic Receptor Functional Potency Assays

A. Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound was determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were rinsed once with dPBS and lifted with Trypsin-EDDA solution(0.05% trypsin/0.53 mm EDDA) as described in the Cell Culture andMembrane Preparation section above. The detached cells were washed twiceby centrifugation at 650×g for five minutes in 50 mLs dPBS. The cellpellet was then re-suspended in 10 mL dPBS, and the cells were countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells were centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶−2.8×10⁶ cells/mL.

The test compound was initially dissolved to a concentration of 400 μMin dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine was diluted in a similarmanner.

To measure oxotremorine inhibition of adenylyl cyclase (AC) activity, 25μL forskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells were added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedAC activity, 25 μL forskolin and oxotremorine (25 μM and 5 μM finalconcentrations, respectively, diluted in dPBS) 25 μL diluted testcompound, and 50 μL cells were added to remaining assay wells.

Reactions were incubated for 10 minutes at 37° C. and stopped byaddition of 100 μL ice-cold detection buffer. Plates were sealed,incubated overnight at room temperature and counted the next morning ona PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) wascalculated based on the counts observed for the samples and cAMPstandards, as described in the manufacturer's user manual. Data wereanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the EC₅₀ of theoxotremorine concentration-response curve and the oxotremorine assayconcentration as the K_(D) and [L], respectively. The K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that a test compound has ahigher functional activity at the receptor tested. The compound ofExample 1 was found to have a K_(i) value of less than 5 nM for blockadeof oxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

B. Blockade of Agonist-Mediated GTPγ[³⁵S] Binding

In a second functional assay, the functional potency of a test compoundis determined by measuring the ability of the compound to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor. At the time of use, frozen membranes were thawed andthen diluted in assay buffer with a final target tissue concentration of5–10 μg protein per well. The membranes were briefly homogenized using aPolytron PT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 uM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that a test compound has ahigher functional activity at the receptor tested. The compound ofExample 1 was found to have a K_(i) value of less than 5 nM for blockadeof oxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

C. Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency is determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stable expressing thehM₁, hM₃ and cM₅ receptors were seeded into 96-well FLIPR plates thenight before the assay was done. Seeded cells were washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mm HEPES, pH 7.4,2 mm calcium chloride, 2.5 mm probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leave 50 μL/well of FLIPR buffer. The cells were then incubated with50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutes at37° C., 5% carbon dioxide. Following the dye incubation period, cellswere washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine was first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells were first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which was performed by the FLIPR. An EC₉₀ value for oxotremorine wasgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100-F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)was prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine was added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR were: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline was determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence was expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data was analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values weredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that a test compound has ahigher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than 5 nM for blockadeof agonist-mediated calcium release in CHO cells stable expressing thehM₃ receptor.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay was used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

The test compound was administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigswere exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols were generated from aqueous solutions using an LC StarNebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian,Va.) driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at apressure of 22 psi. The gas flow through the nebulizer at this operatingpressure was approximately 3 L/minute. The generated aerosols weredriven into the chambers by positive pressure. No dilution air was usedduring the delivery of aerosolized solutions. During the 10 minutenebulization, approximately 1.8 mL of solution was nebulized. This wasmeasured gravimetrically by comparing pre- and post-nebulization weightsof the filled nebulizer.

The bronchoprotective effects of a test compound administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig was anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site was shaved and cleaned with 70% alcohol, a 2–3cm midline incision of the ventral aspect of the neck was made. Then,the jugular vein was isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of acetylcholine (Ach) (Sigma, St. Louis, Mo.)in saline. The trachea was then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia was monitoredand adjusted if the animal responded to pinching of its paw or if therespiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube was attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberwas then sealed. A heating lamp was used to maintain body temperatureand the guinea pig's lungs were inflated 3 times with 4 mL of air usinga 10 mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City,Mo.) to ensure that the lower airways did not collapse and that theanimal did not suffer from hyperventilation.

Once it was determined that baseline values were within the range0.3–0.9 mL/cm H₂O for compliance and within the range 0.1–0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer progam enabled thecollection and derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath were measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowwas calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which were collected using a Sensym pressuretransducer (#TRD4100), was connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with Ach. Ach (Sigma-Aldrich, St. Louis,Mo.) (0.1 mg/mL) was infused intravenously for 1 minute from a syringepump (sp210iw, World Precision Instruments, Inc., Sarasota, Fla.) at thefollowing doses and prescribed times from the start of the experiment:1.9 μg/minute at 5 minutes, 3.8 μg/minute at 10 minutes, 7.5 μg/minuteat 15 minutes, 15.0 μg/minute at 20 minutes, 30 μg/minute at 25 minutesand 60 μg/minute at 30 minutes. If resistance or compliance had notreturned to baseline values at 3 minutes following each Ach dose, theguinea pig's lungs were inflated 3 times with 4 mL of air from a 10 mLcalibration syringe. Recorded pulmonary parameters included respirationfrequency (breaths/minute), compliance (mL/cm H₂O) and pulmonaryresistance (cm H₂O/mL per second). Once the pulmonary functionmeasurements were completed at minute 35 of this protocol, the guineapig was removed from the plethysmograph and euthanized by carbon dioxideasphyxiation.

The data were evaluated in one or both of the following ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) was calculatedfrom the ratio of “change in pressure” to “the change in flow.”

The R_(L) response to ACh (60 μg/min, IH) was computed for the vehicleand the test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, was calculated and used to compute% inhibition of ACh response, at the corresponding pre-treatment time,at each test compound dose. Inhibition dose-response curves for ‘R_(L)’were fitted with a four parameter logistic equation using GraphPadPrism, version 3.00 for Windows (GraphPad Software, San Diego, Calif.)to estimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used was asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))

where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of Ach or histamineneeded to cause a doubling of the baseline pulmonary resistance, wascalculated using the pulmonary resistance values derived from the flowand the pressure over a range of Ach or histamine challenges using thefollowing equation (which is derived from an equation used to calculatePC₂₀ values described in American Thoracic Society. Guidelines formethacholine and exercise challenge testing—1999. Am J Respir Crit CareMed. 2000; 161:309–329):

${PD}_{2} = {{antilog}\left\lbrack {{\log\; C_{1}} + \frac{\left( {{\log\; C_{2}} - {\log\; C_{1}}} \right)\left( {{2R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$

-   -   where:    -   C₁=concentration of Ach or histamine preceding C₂    -   C₂=concentration of Ach or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

An efficacious dose was defined as a dose that limited thebronchorestriction response to a 50 μg/mL dose of Ach to a doubling ofthe baseline pulmonary resistance (PD₂₍₅₀₎).

Statistical analysis of the data was performed using a two-tailedStudents t-test. A P-value <0.05 was considered significant.

Generally, a test compound having a PD₂₍₅₀₎ less than about 200 μg/mLfor ACh-induced bronchoconstriction at 1.5 hours post-dose in this assayis preferred. The compound of formula I was found to have a PD₂₍₅₀₎ lessthan about 200 μg/mL for ACh-induced bronchoconstriction at 1.5 hourspost-dose.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200–350 g wereacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle were dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R+SMolds, San Carlos, Calif.). Test solutions were dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs were restrained in the inhalation chamber for 30minutes. During this time, guinea pigs were restricted to an area ofapproximately 110 sq. cm. This space was adequate for the animals toturn freely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs were exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs were evaluated at 1.5, 6, 12,24, 48, or 72 hrs after treatment.

Guinea pigs were anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals were placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) was inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, s.c.) was administered andthe gauze pad was immediately discarded and replaced by a newpre-weighed gauze pad. Saliva was collected for 10 minutes, at whichpoint the gauze pad was weighed and the difference in weight recorded todetermine the amount of accumulated saliva (in mg). The mean amount ofsaliva collected for animals receiving the vehicle and each dose of testcompound was calculated. The vehicle group mean was considered to be100% salivation. Results were calculated using result means (n=3 orgreater). Confidence intervals (95%) were calculated for each dose ateach time point using two-way ANOVA. This model is a modified version ofthe procedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol, 1996, 24:243–254.

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, was calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data were fitted to a a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The equationused was as follows:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))

where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ was usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred. In this assay, the compound of formula I hadan apparent lung-selectivity index greater than about 5.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g were used in these studies. Underisoflurane anesthesia (to effect), animals were instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The catheterswere exteriorized utilizing a subcutaneous tunnel to the subscapulararea. All surgical incisions were sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal wasadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM). Animals were allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals were weighedand the carotid artery catheter on each animal was connected to atransducer for recording arterial pressure. Arterial pressure and heartrate was recorded using a Biopac MP-100 Acquisition System. Animals wereallowed to acclimate and stabilize for a period of 20 minutes.

Each animal was challenged with methylcholine (MCh) (0.3 mg/kg, iv)administer through the jugular venous line and the cardiovascularresponse was monitored for 10 minutes. The animals were then placed intothe whole body dosing chamber, which was connected to a nebulizercontaining the test compound or vehicle solution. The solution wasnebulized for 10 minutes using a gas mixture of breathable air and 5%carbon dioxide with a flow rate of 3 liters/minute. The animals werethen removed from the whole body chamber and returned to theirrespective cages. At 1.5 and 24 h post-dosing, the animals werere-challenged with MCh (0.3 mg/kg, iv) and the hemodynamic response wasdetermined. Thereafter, the animals were euthanized with sodiumpentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) was measured for each MCh challenge (before andafter IH dosing). The bradycardic effects were not used for analysissince these responses were not robust and reproducible. The effects oftreatment on the MCh responses are expressed as % inhibition(mean+/−SEM) of the control depressor responses. Two-way ANOVA with theappropriate post-hoc test was used to test the effects of treatment andpre-treatment time. The depressor responses to MCh were relativelyunchanged at 1.5 and 24 h after inhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ was usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. In this assay, the compound of Example 1 had an apparentlung-selectivity index greater than 5.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. A compound of formula I:

wherein each R¹ and R² is independently selected from C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₃₋₆ cycloalkyl, cyano, halo, —OR^(a), —SR^(a),—S(O)R^(a), —S(O)₂R^(a) and —NR^(b)R^(c); or two adjacent R¹ groups ortwo adjacent R² groups are joined to form C₃₋₆ alkylene, —(C₂₋₄alkylene)—O— or —O—(C₁₋₄ alkylene)—O—; each R³ is independently selectedfrom C₁₋₄ alkyl and fluoro; each R⁴ is independently selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, C₆₋₁₀aryl, C₂₋₉ heteroaryl, C₃₋₆ heterocyclic, —CH₂—R⁶ and —CH₂CH₂—R⁷; orboth R⁴ groups are joined together with the nitrogen atom to which theyare attached to form C₃₋₆ heterocyclic; R⁵ is selected from C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, and —CH₂—R⁸; wherein eachalkyl, alkenyl and alkynyl group is optionally substituted with —OH or 1to 5 fluoro substituent; each R⁶ is independently selected from C₃₋₆cycloalkyl, C₆₋₁₀ aryl, C₂₋₉ heteroaryl and C₃₋₆ heterocyclic; each R⁷is independently selected from C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl, C₃₋₆ heterocyclic, —OH, —O(C₁₋₆ alkyl), —O(C₃₋₆ cycloalkyl),—O(C₆₋₁₀ aryl), —O(C₂₋₉ heteroaryl), —S(C₁₋₆ alkyl), —S(O)(C₁₋₆ alkyl),—S(O)₂(C₁₋₆ alkyl), —S(C₃₋₆ cycloalkyl), —S(O)(C₃₋₆ cycloalkyl),—S(O)₂(C₃₋₆ cycloalkyl), —S(C₆₋₁₀ aryl), —S(O)(C₆₋₁₀ aryl), —S(O)₂(C₆₋₁₀aryl), —S(C₂₋₉ heteroaryl), —S(O)(C₂₋₉ heteroaryl) and —S(O)₂(C₂₋₉heteroaryl); each R⁸ is independently selected from C₃₋₆ cycloalkyl,C₆₋₁₀ aryl, C₂₋₉ heteroaryl and C₃₋₆ heterocyclic; each R^(a) isindependently selected from hydrogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl and C₃₋₆ cycloalkyl; each R^(b) and R^(c) is independentlyselected from hydrogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl and C₃₋₆cycloalkyl; or R^(b) and R^(c) are joined together with the nitrogenatom to which they are attached to form C₃₋₆ heterocyclic; a is aninteger from 0 to 3; b is an integer from 0 to 3; c is an integer from 0to 4; d is 1 or 2; e is 8 or 9; wherein each alkyl, alkylene, alkenyl,alkynyl and cycloalkyl group in R¹, R², R³, R⁴, R⁷, R^(a), R^(b) andR^(c) is optionally substituted with 1 to 5 fluoro substituent; eacharyl, cycloalkyl, heteroaryl and heterocyclic group in R¹, R², R⁴, R⁵,R⁶, R⁷, R⁸, R^(a), R^(b) and R^(c) is optionally substituted with 1 to 3substituents independently selected from C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl, cyano, halo, —O(C₁₋₄ alkyl), —S(C₁₋₄ alkyl), —S(O)(C₁₋₄ alkyl),—S(O)₂(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl) and —N(C₁₋₄ alkyl)₂, whereineach alkyl, alkylene, alkenyl and alkynyl group is optionallysubstituted with 1 to 5 fluoro substituent; and each —CH₂— group in—(CH₂)_(e)— is optionally substituted with 1 or 2 substituentsindependently selected from C₁₋₂ alkyl, —OH and fluoro; or apharmaceutically-acceptable salt or solvate or stereoisomer thereof. 2.The compound of claim 1, wherein R⁵ is C₁₋₅ alkyl, wherein the alkylgroup is optionally substituted with —OH or 1 to 3 fluoro substituents.3. The compound of claim 2, wherein R⁵ is C₁₋₃ alkyl, wherein the alkylgroup is optionally substituted with —OH or 1 to 3 fluoro substituents.4. The compound of claim 2, wherein R⁵ is selected from methyl, ethyl,2-hydroxyethyl, 2,2,2-trifluoroethyl, n-propyl, isopropyl,1-hydroxyprop-2-yl, n-butyl and isobutyl.
 5. The compound of claim 4,wherein R⁵ is methyl.
 6. The compound of claim 1, wherein R⁵ is C₃₋₅cycloalkyl, wherein the cycloalkyl group is optionally substituted with—OH or 1 to 3 fluoro substituents.
 7. The compound of claim 6, whereinR⁵ is selected from cyclopropyl, cyclobutyl and cyclopentyl.
 8. Thecompound of claim 1, wherein R⁵ is selected from: (a) —CH₂—(C₃₋₅cycloalkyl), wherein the cycloalkyl group is optionally substituted with—OH or 1 to 3 fluoro substituents; and (b) —CH₂—(phenyl), wherein thephenyl group is optionally substituted with 1 to 3 substituentsindependently selected from C₁₋₄ alkyl, cyano, fluoro, chloro, —O(C₁₋₄alkyl), —S(C₁₋₄ alkyl) and —S(O)₂(C₁₋₄ alkyl); where each alkyl group isoptionally substituted with 1 to 3 fluoro substituent.
 9. The compoundof claim 8, wherein R⁵ is selected from cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, benzyl, 4-cyanobenzyl,3-methylbenzyl, 4-methylbenzyl, 4-trifluoromethoxybenzyl, 3-fluorobenzyland 4-fluorobenzyl.
 10. The compound of claim 1, wherein each R⁴ ishydrogen.
 11. The compound of claim 1, wherein a, b and c are
 0. 12. Thecompound of claim 1, wherein d is
 1. 13. The compound of claim 1,wherein d is
 2. 14. The compound of claim 1, wherein e is
 8. 15. Thecompound of claim 1, wherein e is
 9. 16. A compound of formula II:

wherein R⁵ is selected from C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, and —CH₂—R⁸; wherein each alkyl, alkenyl and alkynyl groupis optionally substituted with —OH or 1 to 5 fluoro substituent; each R⁸is independently selected from C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl and C₃₋₆ heterocyclic; e is 8 or 9; wherein each aryl,cycloalkyl, heteroaryl and heterocyclic group in R⁵ and R⁸ is optionallysubstituted with 1 to 3 substituents independently selected from C₁₋₄alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, cyano, halo, —O(C₁₋₄ alkyl), —S(C₁₋₄alkyl), —S(O)(C₁₋₄ alkyl), —S(O)₂(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl) and—N(C₁₋₄ alkyl)₂, wherein each alkyl, alkylene, alkenyl and alkynyl groupis optionally substituted with 1 to 5 fluoro substituent; and each —CH₂—group in —(CH₂)_(e)— is optionally substituted with 1 or 2 substituentsindependently selected from C₁₋₂ alkyl, —OH and fluoro; or apharmaceutically-acceptable salt or solvate or stereoisomer thereof. 17.The compound of claim 16, wherein the stereochemistry at the 3-positionof the pyrrolidine ring is (S).
 18. The compound of claim 16, whereinthe stereochemistry at the 3-position of the pyrrolidine ring is (R).19. The compound of claim 17 or 18, wherein R⁵ is C₁₋₃ alkyl wherein thealkyl group is optionally substituted with —OH or 1 to 3 fluorosubstituents.
 20. The compound of claim 17 or 18, wherein R⁵ is methyl.21. A compound selected from:2-[(S)-1-(8-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-isopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-prop-1-ylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-cyclopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-cyclobutylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-cyclopentylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(8-ethylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(S)-1-[8-(2-hydroxyethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[8-(R)-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[8-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[8-(S)-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[8-(2,2,2-trifluoroethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-[(S)-1-(8-benzylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-isopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-prop-1-ylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-cyclopropylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-cyclobutylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-cyclopentylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(8-ethylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(R)-1-[8-(2-hydroxyethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[8-(R)-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[8-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[8-(S)-(1-hydroxyprop-2-yl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[8-(2,2,2-trifluoroethyl)aminooctyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-[(R)-1-(8-benzylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-methylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-isopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-prop-1-ylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-cyclopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-cyclobutylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-cyclopentylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(9-ethylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(S)-1-[9-(2-hydroxyethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[9-(R)-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[9-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[9-(S)-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[9-(2,2,2-trifluoroethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-[(S)-1-(9-benzylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-methylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-isopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-prop-1-ylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-cyclopropylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-cyclobutylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-cyclopentylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(R)-1-(9-ethylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(R)-1-[9-(2-hydroxyethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[9-(R)-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[9-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[9-(S)-(1-hydroxyprop-2-yl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(R)-1-[9-(2,2,2-trifluoroethyl)aminononyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-[(R)-1-(9-benzylaminononyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[1-(8-methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[1-(8-isopropylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(8-prop-1-ylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(8-cyclopropylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(8-cyclobutylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(8-cyclopentylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(8-ethylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-{1-[8-(2-hydroxyethyl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[8-(R)-(1-hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[8-(1-hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[8-(S)-(1-hydroxyprop-2-yl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[8-(2,2,2-trifluoroethyl)aminooctyl]piperidin-4-yl}-2,2-diphenylacetamide;2-[1-(8-benzylaminooctyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-methylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-isopropylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-prop-1-ylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-cyclopropylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-cyclobutylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-cyclopentylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-[1-(9-ethylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide;2-{1-[9-(2-hydroxyethyl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[9-(R)-(1-hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[9-(1-hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[9-(S)-(1-hydroxyprop-2-yl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide;2-{1-[9-(2,2,2-trifluoroethyl)aminononyl]piperidin-4-yl}-2,2-diphenylacetamide;and 2-[1-(9-benzylaminononyl)piperidin-4-yl]-2,2-diphenylacetamide; or apharmaceutically-acceptable salt or solvate thereof. 22.2-[(S)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide or apharmaceutically-acceptable salt or solvate thereof. 23.2-[(R)-1-(8-Methylaminooctyl)pyrrolidin-3-yl]-2,2-diphenylacetamide or apharmaceutically-acceptable salt or solvate thereof.